Microorganisms and methods for conversion of syngas and other carbon sources to useful products

ABSTRACT

A non-naturally occurring microbial organism having an isopropanol, 4-hydroxybutryate, or 1,4-butanediol pathway includes at least one exogenous nucleic acid encoding an isopropanol, 4-hydroxybutryate, or 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce isopropanol, 4-hydroxybutryate, or 1,4-butanediol. The aforementioned organisms are cultured to produce isopropanol, 4-hydroxybutryate, or 1,4-butanediol.

This application is a continuation-in-part of PCT Application NumberPCT/US09/50757, filed Jul. 15, 2009 and further claims the benefit ofpriority of U.S. Provisional Application Ser. No. 61/138,108, filed Dec.16, 2008, each of which the entire contents are incorporated herein bythis reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes andorganisms capable of converting carbohydrates, methanol, synthesis gasand other gaseous carbon sources into higher-value chemicals.

Increasing the flexibility of cheap and readily available feedstocks andminimizing the environmental impact of chemical production arebeneficial for a sustainable chemical industry. Feedstock flexibilityrelies on the introduction of methods that can access and use a widerange of materials as primary feedstocks for chemical manufacturing.

Isopropanol (IPA) is a colorless, flammable liquid that mixes completelywith most solvents, including water. The largest use for IPA is as asolvent, including its well known yet small use as “rubbing alcohol,”which is a mixture of IPA and water. As a solvent, IPA is found in manyeveryday products such as paints, lacquers, thinners, inks, adhesives,general-purpose cleaners, disinfectants, cosmetics, toiletries,de-icers, and pharmaceuticals. Low-grade IPA is also used in motor oils.The second largest use is as a chemical intermediate for the productionof isopropylamines (e.g. in agricultural products), isopropylethers, andisopropyl esters.

Isopropanol is manufactured by two petrochemical routes. The predominantprocess entails the hydration of propylene either with or withoutsulfuric acid catalysis. Secondarily, IPA is produced via hydrogenationof acetone, which is a by-product formed in the production of phenol andpropylene oxide. High-priced propylene is currently driving costs up andmargins down throughout the chemical industry motivating the need for anexpanded range of low cost feedstocks.

4-hydroxybutanoic acid (4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB) isa 4-carbon carboxylic acid that has industrial potential as a buildingblock for various commodity and specialty chemicals. In particular, 4-HBhas the potential to serve as a new entry point into the 1,4-butanediolfamily of chemicals, which includes solvents, resins, polymerprecursors, and specialty chemicals.

BDO is a valuable chemical for the production of high performancepolymers, solvents, and fine chemicals. It is the basis for producingother high value chemicals such as tetrahydrofuran (THF) andgamma-butyrolactone (GBL). Uses of BDO include (1) polymers, (2) THFderivatives, and (3) GBL derivatives. In the case of polymers, BDO is aco-monomer for polybutylene terephthalate (PBT) production. PBT is amedium performance engineering thermoplastic made by companies such asDuPont and General Electric finding use in automotive, electrical, watersystems, and small appliance applications. When converted to THF, andsubsequently to polytetramethylene ether glycol (PTMEG), the Spandex andLycra fiber and apparel industries are added to the markets served.PTMEG is also combined with BDO in the production of specialty polyesterethers (COPE). COPEs are high modulus elastomers with excellentmechanical properties and oil/environmental resistance, allowing them tooperate at high and low temperature extremes. PTMEG and BDO also makethermoplastic polyurethanes processed on standard thermoplasticextrusion, calendaring, and molding equipment, and are characterized bytheir outstanding toughness and abrasion resistance. The GBL producedfrom BDO provides the feedstock for making pyrrolidones, as well asserving agrochemical market applications itself. The pyrrolidones areused as high performance solvents for extraction processes of increasinguse in the electronics industry as well as use in pharmaceuticalproduction.

BDO is produced by two main petrochemical routes with a few additionalroutes also in commercial operation. One route involves reactingacetylene with formaldehyde, followed by hydrogenation. More recentlyBDO processes involving butane or butadiene oxidation to maleicanhydride, followed by hydrogenation have been introduced. BDO is usedalmost exclusively as an intermediate to synthesize other chemicals andpolymers.

Synthesis gas (syngas) is a mixture of primarily H₂ and CO that can beobtained via gasification of any organic feedstock, such as coal, coaloil, natural gas, biomass, or waste organic matter. Numerousgasification processes have been developed, and most designs are basedon partial oxidation, where limiting oxygen avoids full combustion, oforganic materials at high temperatures (500-1500° C.) to provide syngasas, for example, 0.5:1-3:1 H₂/CO mixture. Steam is sometimes added toincrease the hydrogen content, typically with increased CO₂ productionthrough the water gas shift reaction. Methanol is most commonly producedindustrially from the syngas components, CO and H₂, via catalysis.

Today, coal is the main substrate used for industrial production ofsyngas, which is usually used for heating and power and as a feedstockfor Fischer-Tropsch synthesis of methanol and liquid hydrocarbons. Manylarge chemical and energy companies employ coal gasification processeson large scale and there is experience in the industry using thistechnology.

Overall, technology now exists for cost-effective production of syngasfrom a plethora of materials, including coal, biomass, wastes, polymers,and the like, at virtually any location in the world. Biomassgasification technologies are being practiced commercially, particularlyfor heat and energy generation.

Despite the availability of organisms that utilize syngas, suchorganisms are generally poorly characterized and are not well-suited forcommercial development. For example, Clostridium and related bacteriaare strict anaerobes that are intolerant to high concentrations ofcertain products such as butanol, thus limiting titers andcommercialization potential. The Clostridia also produce multipleproducts, which presents separations issues in isolating a desiredproduct. Finally, development of facile genetic tools to manipulateclostridial genes is in its infancy, therefore, they are not currentlyamenable to rapid genetic engineering to improve yield or productioncharacteristics of a desired product.

Thus, there exists a need to develop microorganisms and methods of theiruse to utilize carbohydrates, methanol, syngas and/or other gaseouscarbon sources for the production of desired chemicals and fuels. Morespecifically, there exists a need to develop microorganisms forcarbohydrate, methanol, and syngas utilization that also have existingand efficient genetic tools to enable their rapid engineering to producevaluable products at useful rates and quantities. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

In some aspects, the present invention provides a non-naturallyoccurring microbial organism having an isopropanol pathway that includesat least one exogenous nucleic acid encoding an isopropanol pathwayenzyme expressed in a sufficient amount to produce isopropanol. Theisopropanol pathway enzyme includes a succinyl-CoA:3-ketoacid-CoAtransferase.

In other aspects, the present invention provides a non-naturallyoccurring microbial organism having a 4-hydroxybutryate pathway thatincludes at least one exogenous nucleic acid encoding an4-hydroxybutryate pathway enzyme expressed in a sufficient amount toproduce 4-hydroxybutryate. The 4-hydroxybutryate pathway enzyme includesan acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, acrotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA transferase,a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, aphosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase.

In still other aspects, the present invention provides a non-naturallyoccurring microbial organism having a 1,4-butanediol pathway thatincludes at least one exogenous nucleic acid encoding a 1,4-butanediolpathway enzyme expressed in a sufficient amount to produce1,4-butanediol. The 1,4-butanediol pathway enzyme includes anacetoacetyl-CoA thiolase, a 3-Hydroxybutyryl-CoA dehydrogenase, acrotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase(alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming),a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, aphosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a4-hydroxybutyrate reductase. Such an organism also includes anacetyl-CoA pathway having at least one exogenous nucleic acid encodingan acetyl-CoA pathway enzyme expressed in a sufficient amount to produceacetyl-CoA. The acetyl-CoA pathway enzyme includes a corrinoid protein,a methyltetrahydrofolate:corrinoid protein methyltransferase, acorrinoid iron-sulfur protein, a nickel-protein assembly protein, aferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, apyruvate ferredoxin oxidoreductase, and a hydrogenase.

In yet other aspects, the present invention provides a non-naturallyoccurring microbial organism having a 1,4-butanediol pathway thatincludes at least one exogenous nucleic acid encoding a 1,4-butanediolpathway enzyme expressed in a sufficient amount to produce1,4-butanediol. The 1,4-butanediol pathway enzyme includes anacetoacetyl-CoA thiolase, a 3-Hydroxybutyryl-CoA dehydrogenase, acrotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase(alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming),a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, aphosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a4-hydroxybutyrate reductase. Such an organism also includes anacetyl-CoA pathway having at least one exogenous nucleic acid encodingan acetyl-CoA pathway enzyme expressed in a sufficient amount to produceacetyl-CoA. The acetyl-CoA pathway enzyme includes an acetyl-CoAsynthase, a formate dehydrogenase, a formyltetrahydrofolate synthetase,a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase.

In still further aspects, the present invention provides a non-naturallyoccurring microbial organism having an isopropanol pathway that includesat least one exogenous nucleic acid encoding an isopropanol pathwayenzyme expressed in a sufficient amount to produce isopropanol. Theisopropanol pathway enzyme includes an acetoacetyl-CoA thiolase, anacetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, anacetoacetyl-CoA synthetase, a phosphotransacetoacetylase, anacetoacetate kinase, an acetoacetate decarboxylase, and an isopropanoldehydrogenase. Such an organism also includes at least one exogenousnucleic acid encoding an acetyl-CoA enzyme expressed in a sufficientamount to produce acetyl-CoA. The acetyl-CoA pathway enzyme includes amethanol methyl transferase, a corrinoid protein, amethyltetrahydro-folate:corrinoid protein methyltransferase, a corrinoidiron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, anacetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvateferredoxin oxidoreductase, and a hydrogenase.

In still further aspects, the present invention provides a method forproducing isopropanol that includes culturing a non-naturally occurringmicrobial organism having an isopropanol pathway. The pathway includesat least one exogenous nucleic acid encoding an isopropanol pathwayenzyme expressed in a sufficient amount to produce isopropanol underconditions and for a sufficient period of time to produce isopropanol.The isopropanol pathway includes a succinyl-CoA:3-ketoacid-CoAtransferase.

In yet still further aspects, the present invention provides a methodfor producing 4-hydroxybutyrate that includes culturing a non-naturallyoccurring microbial organism having an 4-hydroxybutyrate pathway. Thepathway includes at least one exogenous nucleic acid encoding an4-hydroxybutyrate pathway enzyme expressed in a sufficient amount toproduce 4-hydroxybutyrate under conditions and for a sufficient periodof time to produce 4-hydroxybutyrate. The 4-hydroxybutyrate pathwayincludes an acetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoAdehydrogenase, a crotonase, a crotonyl-CoA hydratase, a4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a4-hydroxybutyryl-CoA synthetase, a phosphotrans-4-hydroxybutyrylase, anda 4-hydroxybutyrate kinase.

In still other aspects, the present invention provides a method forproducing 1,4-butanediol that includes culturing a non-naturallyoccurring microbial organism having an 1,4-butanediol pathway. Thepathway includes at least one exogenous nucleic acid encoding an1,4-butanediol pathway enzyme expressed in a sufficient amount toproduce 1,4-butanediol under conditions and for a sufficient period oftime to produce 1,4-butanediol. The 1,4-butanediol pathway includes anacetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, acrotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase(alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming),a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, aphosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a4-hydroxybutyrate reductase. Such an organism also includes anacetyl-CoA pathway comprising at least one exogenous nucleic acidencoding an acetyl-CoA pathway enzyme expressed in a sufficient amountto produce acetyl-CoA. The acetyl-CoA pathway enzyme includes acorrinoid protein, a methyltetrahydro-folate:corrinoid proteinmethyltransferase, a corrinoid iron-sulfur protein, a nickel-proteinassembly protein, a ferredoxin, an acetyl-CoA synthase, a carbonmonoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and ahydrogenase.

Finally, in some aspects, the present invention provides a method forproducing 1,4-butanediol that includes culturing a non-naturallyoccurring microbial organism having an 1,4-butanediol pathway. Thepathway includes at least one exogenous nucleic acid encoding an1,4-butanediol pathway enzyme expressed in a sufficient amount toproduce 1,4-butanediol under conditions and for a sufficient period oftime to produce 1,4-butanediol. The 1,4-butanediol pathway includes anacetoacetyl-CoA thiolase, a 3-hydroxybutyryl-CoA dehydrogenase, acrotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase(alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming),a 1,4-butanediol dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, aphosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a4-hydroxybutyrate reductase. Such an organism also includes anacetyl-CoA pathway having at least one exogenous nucleic acid encodingan acetyl-CoA pathway enzyme expressed in a sufficient amount to produceacetyl-CoA. The acetyl-CoA pathway enzyme includes an acetyl-CoAsynthase, a formate dehydrogenase, a formyltetrahydrofolate synthetase,a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram depicting the Wood-Ljungdahl pathway andformation routes for acetate and ethanol. The transformations that aretypically unique to organisms capable of growth on synthesis gas are 1)CO dehydrogenase, 2) hydrogenase, 3) energy-conserving hydrogenase(ECH), and 4) bi-functional CO dehydrogenase/acetyl-CoA synthase.Oxidation of hydrogen to 2 [H] or of CO with H₂O to CO₂ and 2 [H]provides reducing equivalents for the reduction of CO₂ to formate, ofmethenyl-tetrahydrofolate (methenyl-THF) to methylene-tetrahydrofolate(methylene-THF), of methylene-THF to methyltetrahydrofolate(methyl-THF), and of CO₂ to CO.

FIG. 2A shows the complete Wood-Ljungdahl pathway for the conversion ofcarbohydrates and/or gases including CO, CO₂, and/or H₂ to acetyl-CoAwhich is subsequently converted to cell mass and products such asethanol or acetate. FIG. 2B shows a synthetic metabolic pathway for theconversion of carbohydrates and/or gases including CO, CO₂, and/or H₂,and methanol to acetyl-CoA and further to isopropanol. FIG. 2C shows asynthetic metabolic pathway for the conversion of carbohydrates and/orgases including CO, CO₂, and/or H₂, and methanol to acetyl-CoA andfurther to 4-hydroxybutyrate. FIG. 2D shows a synthetic metabolicpathway for the conversion of carbohydrates and/or gases including CO,CO₂, and/or H₂, and methanol to acetyl-CoA and further to1,4-butanediol. In FIGS. 2A-D, the specific enzymatic transformationsthat can be engineered into a production host are numbered.Abbreviations: 10FTHF: 10-formyltetrahydrofolate, 5MTHF:5-methyltetrahydrofolate, ACTP: acetyl phosphate, CFeSp: corrinoid ironsulfur protein, FOR: formate, MeOH: methanol, METHF:methyltetrahydrofolate, MLTHF: metheneyltetrahydrofolate, THF:tetrahydrofolate.

FIG. 3A shows a synthetic metabolic pathway for the conversion ofcarbohydrates and/or gases including CO, CO₂, and/or H₂ to acetyl-CoA,and further to isopropanol. FIG. 3B shows a synthetic metabolic pathwayfor the conversion of carbohydrates and/or gases including CO, CO₂,and/or H₂ to acetyl-CoA, and further to 4-hydroxybutyrate. FIG. 3C showsa synthetic metabolic pathway for the conversion of carbohydrates and/orgases including CO, CO₂, and/or H₂ to acetyl-CoA, and further to1,4-butanediol. In FIGS. 3A-C the specific enzymatic transformationsthat can be engineered into a production host are numbered.Abbreviations: 10FTHF: 10-formyltetrahydrofolate, 5MTHF:5-methyltetrahydrofolate, ACTP: acetyl phosphate, CFeSp: corrinoid ironsulfur protein, FOR: formate, MeOH: methanol, METHF:methyltetrahydrofolate, MLTHF: metheneyltetrahydrofolate, THF:tetrahydrofolate.

FIG. 4 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91(lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards(lane 5) and controls of M. thermoacetica CODH (Moth_(—)1202/1203) orMtr (Moth_(—)1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and1000 ng).

FIG. 5 shows cuvettes used in a methyl viologen assay. A blank is on theright and a cuvette with reduced methyl viologen is on the left.Stoppers and vacuum grease on top of each are used to keep the reactionsanaerobic.

FIG. 6 shows a spectrogram of ACS90 cell extracts assayed for transferof CH₃ from added CH₃-THF to purified M. thermoacetica corrinoidprotein.

FIG. 7 shows anaerobic growth of recombinant E. coli MG1655 in N₂ and COfor 36 hr at 37° C. From left to right: Empty vector, ACS90, and ACS91are shown.

FIG. 8A shows a synthetic metabolic pathway and exemplary fluxdistribution for the conversion of carbohydrates and CO₂ to acetyl-CoA,and further to isopropanol. FIG. 8B shows a synthetic metabolic pathwayand exemplary flux distribution for the conversion of carbohydrates andCO₂ to acetyl-CoA, and further to 4-hydroxybutyrate. FIG. 8C shows asynthetic metabolic pathway and exemplary flux distribution for theconversion of carbohydrates and CO₂ to acetyl-CoA, and further to1,4-butanediol. In FIGS. 8A-C, several enzymatic transformations thatcan be engineered into a production host are numbered.

FIG. 9 shows a synthetic metabolic pathway and exemplary fluxdistribution for the conversion of carbohydrates, methanol and CO₂ toacetyl-CoA, and further to isopropanol. Several enzymatictransformations that can be engineered into a production host arenumbered.

FIG. 10 plots the maximum yield of isopropanol or 4-hydroxybutyrate as afunction of the glucose:methanol ratio.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed, in part, to non-naturally occurringmicroorganisms that express genes encoding enzymes that catalyze thecarbonyl-branch of the Wood-Ljungdahl pathway in conjunction with aMtaABC-type methyltransferase system. Such organisms are capableconverting carbohydrates, methanol, a relatively inexpensive organicfeedstock that can be derived from synthesis gas, and gasses includingCO, CO₂, and/or H₂ into acetyl-CoA, cell mass, and products such asisopropanol (IPA), 4-hydroxybutyrate (4-HB), and 1,4-butanediol (BDO).Additional product molecules that can be produced by the teachings ofthis invention include but are not limited to ethanol, butanol,isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaric acid,malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid,adipic acid, 6-aminocaproic acid, hexamethylenediamine,3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid,acrylic acid, and long chain hydrocarbons, alcohols, acids, and esters.The invention is also directed, in part, to non-naturally occurringmicroorganisms that express genes encoding enzymes that catalyze thecarbonyl and methyl-branches of the Wood-Ljungdahl pathway. Suchorganisms are capable converting gasses including CO, CO₂, and/or H₂into acetyl-CoA, cell mass, and products such as IPA, 4-HB, and BDO.Additional product molecules include but are not limited to ethanol,butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaricacid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lacticacid, adipic acid, 6-aminocaproic acid, hexamethylenediamine,3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid,acrylic acid, and long chain hydrocarbons, alcohols, acids, and esters.

In one embodiment, the invention provides non-naturally occurringmicrobial organisms capable of producing isopropanol, 4-hydroxybutyrate,or 1,4-butanediol from methanol and gaseous feedstocks such as mixturesof syngas. In other embodiments, the invention provides non-naturallyoccurring microbial organisms capable of producing isopropanol,4-hydroxybutyrate, or 1,4-butanediol from mixtures of syngas, withoutthe need for methanol. In further embodiments, the invention providespathways for increased yield of isopropanol, 4-hydroxybutyrate or1,4-butanediol on carbohydrate feedstocks over what would be naturallyexpected (e.g., one mol isopropanol, 4-hydroxybutyrate or 1,4-butanediolper one mole glucose consumed) by providing an efficient mechanism forfixing the carbon present in methanol or carbon dioxide, fedexogeneously or produced endogenously, into acetyl-CoA.

In another embodiment, the invention provides methods for producingisopropanol, 4-hydroxybutyrate, or 1,4-butanediol through culturing ofthese non-naturally occurring microbial organisms. Biotechnologicalprocesses utilizing these organisms will provide operational flexibilityfor isopropanol, 4-hydroxybutyrate, and 1,4-butanediol manufacturers toassure the lowest operating costs based on diversified feedstocks andoptionally to offset volatility in market-driven commodity pricing ofcurrent feedstocks such as oil and natural gas. Furthermore, theseprocesses will deliver sustainable manufacturing practices that utilizerenewable feedstocks, reduce energy intensity and lower greenhouse gasemissions.

Acetogens, such as Moorella thermoacetica, C. ljungdahlii and C.carboxidivorans, can grow on a number of carbon sources ranging fromhexose sugars to carbon monoxide. Hexoses, such as glucose, aremetabolized first via Embden-Meyerhof-Parnas (EMP) glycolysis topyruvate, which is then converted to acetyl-CoA via pyruvate:ferredoxinoxidoreductase (PFOR). Acetyl-CoA can be used to build biomassprecursors or can be converted to acetate which produces energy viaacetate kinase and phosphotransacetylase. The overall conversion ofglucose to acetate, energy, and reducing equivalents is given byequation 1:

C₆H₁₂O₆+4 ADP+4 Pi→2 CH₃COOH+2 CO₂+4 ATP+8 [H]  equation 1

Acetogens extract even more energy out of the glucose to acetateconversion while also maintaining redox balance by further convertingthe released CO₂ to acetate via the Wood-Ljungdahl pathway

2 CO₂+8 [H]+n ADP+n Pi→CH₃COOH+n ATP  equation 2

The coefficient n in the above equation signify that this conversion isan energy generating endeavor, as many acetogens can grow in thepresence of CO₂ via the Wood-Ljungdahl pathway even in the absence ofglucose as long as hydrogen is present to supply the necessary reducingequivalents.

2 CO₂+4H₂ +n ADP+n Pi→CH₃COOH+2H₂O+n ATP  equation 3

The Wood-Ljungdahl pathway, illustrated in FIG. 1, is coupled to thecreation of Na⁺ or H⁺ ion gradients that can generate ATP via an Na⁺- orH⁺-dependant ATP synthase, respectively (Muller, V. Appl EnvironMicrobiol 69:6345-6353 (2003)). Based on these known transformations,acetogens also have the capacity to utilize CO as the sole carbon andenergy source. Specifically, CO can be oxidized to produce reducingequivalents and CO₂, or directly assimilated into acetyl-CoA which issubsequently converted to either biomass or acetate.

4 CO+2H₂O→CH₃COOH+2 CO₂  equation 4

Even higher acetate yields, however, can be attained when enoughhydrogen is present to satisfy the requirement for reducing equivalents.

2 CO+2H₂→CH₃COOH  equation 5

Following from FIG. 1, the production of acetate via acetyl-CoAgenerates one ATP molecule, whereas the production of ethanol fromacetyl-CoA does not and requires two reducing equivalents. Thus onemight speculate that ethanol production from syngas will not generatesufficient energy for cell growth in the absence of acetate production.However, under certain conditions, Clostridium ljungdahlii producesmostly ethanol from synthesis gas (Klasson et al., Fuel 72:1673-1678(1993)). indicating that some combination of the pathways

2 CO₂+6H₂→CH₃CH₂OH+3H₂O  equation 6

6 CO+3H₂O→CH₃CH₂OH+4 CO₂  equation 7

2 CO+4H₂→CH₃CH₂OH+H₂O  equation 8

does indeed generate enough energy to support cell growth. Hydrogenicbacteria such as R. rubrum can also generate energy from the conversionof CO and water to hydrogen (see FIG. 1) (Simpma et al., CriticalReviews in Biotechnology 26:41-65 (2006)). One important mechanism isthe coordinated action of an energy converting hydrogenase (ECH) and COdehydrogenase. The CO dehydrogenase supplies electrons from CO which arethen used to reduce protons to H₂ by ECH, whose activity is coupled toenergy-generating proton translocation. The net result is the generationof energy via the water-gas shift reaction.

Embodiments of the present invention describe the combination of (1)pathways for the conversion of synthesis gases including CO₂, CO, and H₂with and without methanol to acetyl-CoA and (2) pathways for theconversion of acetyl-CoA to isopropanol, 4-hydroxybutyrate, or1,4-butanediol. As such, this invention provides production organismsand conversion routes with inherent yield advantages over organismsengineered to produce isopropanol, 4-hydroxybutyrate, or 1,4-butanediolfrom carbohydrate feedstocks using only the product pathways proceedingfrom acetyl-CoA (i.e., (2) of the combination described above). Forexample, the maximum theoretical yields of isopropanol,4-hydroxybutyrate, and 1,4-butanediol from glucose are 1 mole per moleusing only the metabolic pathways proceeding from acetyl-CoA asdescribed herein. Specifically, 2 moles of acetyl-CoA are derived permole of glucose via glycolysis and 2 moles of acetyl-CoA are requiredper mole of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. The netconversions are described by the following stoichiometric equations:

Isopropanol: C₆H₁₂O₆+1.5 O₂→C₃H₈O+3 CO₂+2H₂O

4-Hydroxybutyate: C₆H₁₂O₆+1.5 O₂→C₄H₈O₃+2 CO₂+2H₂O

1,4-Butanediol: C₆H₁₂O₆→C₄H₁₀O₂+CH₂O₂+CO₂

On the other hand, gasification of glucose to its simpler components, COand H₂, followed by their conversion to isopropanol, 4-hydroxybutyrate,and 1,4-butanediol using the pathways described herein results in thefollowing maximum theoretical yields:

Isopropanol: 6 CO+6H₂→1.333 C₃H₈O+2 CO₂+0.667H₂O

4-Hydroxybutyate: 6 CO+6H₂→1.333 C₄H₈O₃+0.667 CO₂+0.667H₂O

1,4-Butanediol: 6 CO+6H₂→1.091 C₄H₁₀O₂+1.636 CO₂+0.545H₂O

Note that the gasification of glucose can at best provide 6 moles of COand 6 moles of H₂. Similarly, the combination of certainsyngas-utilization pathway components with the acetyl-CoA toisopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathways results inhigh yields of these products from carbohydrates by providing anefficient mechanism for fixing the carbon present in carbon dioxide, fedexogeneously or produced endogenously, into acetyl-CoA (see below andalso Example II).

Isopropanol: C₆H₁₂O₆→1.333 C₃H₈O+2 CO₂+0.667H₂O

4-Hydroxybutyate: C₆H₁₂O₆→1.333 C₄H₈O₃+0.667 CO₂+0.667H₂O

1,4-Butanediol: C₆H₁₂O₆→1.091 C₄H₁₀O₂+1.636 CO₂+0.545H₂O

The maximum theoretical yields of isopropanol, 4-hydroxybutyrate, and1,4-butanediol from synthesis gases or carbohydrates can be furtherenhanced by the addition of methanol as described below:

Isopropanol: CH₄O+6 CO+6H₂→1.667 C₃H₈O+2 CO₂+1.333H₂O

4-Hydroxybutyate: CH₄O+6 CO+6H₂→1.667C₄H₈O₃+0.333 CO₂+1.333H₂O

1,4-Butanediol: CH₄O+6 CO+6H₂→1.364 C₄H₁₀O₂+1.545 CO₂+1.182H₂O

Isopropanol: 2 CH₄O+6 CO+6H₂→2 C₃H₈O+2 CO₂+2H₂O

4-Hydroxybutyate: 2 CH₄O+6 CO+6H₂→2 C₄H₈O₃+2H₂O

1,4-Butanediol: 2 CH₄O+6 CO+6H₂→1.636 C₄H₁₀O₂+1.455 CO₂+1.818H₂O

Isopropanol: CH₄O+C₆H₁₂O₆→1.667 C₃H₈O+2 CO₂+1.333H₂O

4-Hydroxybutyate: CH₄O+C₆H₁₂O₆→1.667 C₄H₈O₃+0.333 CO₂+1.333H₂O

1,4-Butanediol: CH₄O+C₆H₁₂O₆→1.364 C₄H₁₀O₂+1.545 CO₂+1.182H₂O

Isopropanol: 2 CH₄O+C₆H₁₂O₆→2 C₃H₈O+2 CO₂+2H₂O

4-Hydroxybutyate: 2 CH₄O+C₆H₁₂O₆→2 C₄H₈O₃+2H₂O

1,4-Butanediol: 2 CH₄O+C₆H₁₂O₆→1.636 C₄H₁₀O₂+1.455 CO₂+1.818H₂O

Thus, the organisms and conversion routes described herein provide anefficient means of converting carbohydrates to products such asisopropanol, 4-hydroxybutyrate, or 1,4-butanediol. Additional productmolecules that can be produced by the teachings of this inventioninclude but are not limited to ethanol, butanol, isobutanol,isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid,4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipicacid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyricacid, 2-hydroxyisobutyric acid, methacrylic acid, acrylic acid, and longchain hydrocarbons, alcohols, acids, and esters.

In some embodiments, the non-naturally occurring organisms of thepresent invention incorporating the Wood-Ljungdahl pathway can functionin the presence of an electron acceptor other than CO₂. Wood-Ljungdahlpathway enzymes are oxygen sensitive which can preclude the use ofoxygen to improve the energetics of the organism. Under certainconditions, electron acceptor nitrate can block carbon assimilation bythe Wood-Ljungahl pathway, by transcriptional regulation of genesencoding Wood-Ljungahl. Nonetheless, in some embodiments, anon-naturally occurring organism of the invention utilizing theWood-Ljungdahl pathway can use nitrate as an electron acceptor toimprove overall energetics, as described further below.

In addition to challenges in energy balance, a further challenge ofincorporating the Wood-Ljungdahl pathway involves redox balance.Acetogens naturally make acetate as the sole product, not isopropanol.Some syngas utilizers such as Clostridium carboxydivorans have beenshown to make multiple products (i.e., ethanol, butanol, acetate, andbutyrate). Converting 2 acetyl-CoA molecules to one isopropanol provides1 reducing equivalent. By contrast, the aforementioned products canproduce 2 or more reducing equivalents.

Non-naturally occurring organisms of the present invention employing thecombination of pathways disclosed herein can provide a yield ofisopropanol, for example, on glucose up to 0.44 g/g glucose. This yieldis improved over organism that do not incorporate the Wood-Ljungdahlpathway.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within anisopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having isopropanol,4-hydroxybutyrate, or 1,4-butanediol biosynthetic capability, thoseskilled in the art will understand with applying the teaching andguidance provided herein to a particular species that the identificationof metabolic modifications can include identification and inclusion orinactivation of orthologs. To the extent that paralogs and/ornonorthologous gene displacements are present in the referencedmicroorganism that encode an enzyme catalyzing a similar orsubstantially similar metabolic reaction, those skilled in the art alsocan utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

Escherichia coli is a common organism with a well-studied set ofavailable genetic tools. Engineering the capability to convert CO₂, COand/or H₂ into acetyl-CoA, the central metabolite from which cell masscomponents and many valuable products can be derived, into a foreignhost such as E. coli can be accomplished following the expression ofexogenous genes that encode various proteins of the Wood-Ljungdahlpathway. This pathway is active in acetogenic organisms such as Moorellathermoacetica (formerly, Clostridium thermoaceticum), which has been themodel organism for elucidating the Wood-Ljungdahl pathway since itsisolation in 1942 (Fontaine et al., J. Bacteriol. 43:701-715 (1942)).The Wood-Ljungdahl pathway includes two branches: the Eastern (ormethyl) branch that enables the conversion of CO₂ tomethyltetrahydrofolate (Me-THF) and the Western (or carbonyl) branchthat enables the conversion of methyl-THF, CO, and Coenzyme-A intoacetyl-CoA as shown in FIG. 2A. Such an organism is capable ofconverting gases comprising CO, CO₂, and/or H₂ into acetyl-CoA, cellmass, and products such as isopropanol, 4-hydroxybutyrate and1,4-butanediol. Such an organism is also capable of producingisopropanol, 4-hydroxybutyrate or 1,4-butanediol from carbohydrates atthe stoichiometric optimum yield. For example, in combination with theacetyl-CoA to isopropanol pathway, the Wood-Ljungdahl enzymes providethe means to produce 4 moles of isopropanol for every 3 moles of glucoseas opposed to 1 mol isopropanol/1 mol glucose which would be attainablein the absence of the Wood-Ljungdahl pathway enzymes. In someembodiments, the present invention provides a non-naturally occurringmicroorganism expressing genes encoding enzymes that catalyze thecarbonyl-branch of the Wood-Ljungdahl pathway in conjunction with aMtaABC-type methyltransferase system. Such an organism is capableconverting methanol, a relatively inexpensive organic feedstock that canbe derived from synthesis gas, and gases including CO, CO₂, and/or H₂into acetyl-CoA, cell mass, and products. In addition to gaseoussubstrates, the organism can utilize methanol exclusively or incombination with carbohydrate feedstocks such as glucose to produceproducts such as isopropanol, 4-hydroxybutyrate or 1,4-butanediol in ahigh yield. Additional product molecules that can be produced by theteachings of this invention include but are not limited to ethanol,butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaricacid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lacticacid, adipic acid, 6-aminocaproic acid, hexamethylenediamine,3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid,acrylic acid, and long chain hydrocarbons, alcohols, acids, and esters.

In some embodiments organisms of the present invention have thefollowing capabilities as depicted in FIG. 2B: 1) a functionalmethyltransferase system enabling the production of5-methyl-tetrahydrofolate (Me-THF) from methanol and THF, 2) the abilityto combine CO, Coenzyme A, and the methyl group of Me-THF to formacetyl-CoA, and 3) the ability to synthesize IPA from acetyl-CoA. Inother embodiments, organisms of the present invention have a functionalmethyltransferase system, the ability to synthesize acetyl-CoA, and theability to synthesize 4-HB from acetyl-CoA as depicted in FIG. 2C. Stillother organisms described herein have a functional methyltransferasesystem, the ability to synthesize acetyl-CoA, and the ability tosynthesize BDO from acetyl-CoA depicted in FIG. 2D.

In some embodiments, organisms of the present invention are able to‘fix’ carbon from exogenous CO and/or CO₂ and methanol to synthesizeacetyl-CoA, cell mass, and products. The direct conversion of synthesisgas to acetate is an energetically neutral process (see FIGS. 1 and 2A).Specifically, one ATP molecule is consumed during the formation offormyl-THF by formyl-THF synthase and one ATP molecule is producedduring the production of acetate via acetate kinase. ATP consumption iscircumvented by ensuring that the methyl group on the methyl branchproduct, methyl-THF, is obtained from methanol rather than CO₂. Thisthereby ensures that acetate formation has a positive ATP yield that canhelp support cell growth and maintenance. A host organism engineeredwith these capabilities that also naturally possesses the capability foranapleurosis, such as E. coli, can grow on the methanol andsyngas-generated acetyl-CoA in the presence of a suitable externalelectron acceptor such as nitrate. This electron acceptor is used toaccept electrons from the reduced quinone formed via succinatedehydrogenase. One advantage of adding an external electron acceptor isthat additional energy for cell growth, maintenance, and productformation can be generated from respiration of acetyl-CoA. In otherembodiments, a pyruvate ferredoxin oxidoreductase (PFOR) enzyme can beinserted into the strain to provide the synthesis of biomass precursorsin the absence of an external electron acceptor. A furthercharacteristic of organisms of the present invention is the capabilityof extracting reducing equivalents from molecular hydrogen. This enablesa high yield of reduced products such as ethanol, butanol, isobutanol,isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid,4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipicacid, 6-aminocaproic acid, hexamethylenediamine, 3-hydroxyisobutyricacid, 2-hydroxyisobutyric acid, methacrylic acid, and acrylic acid.

Organisms of the present invention can produce acetyl-CoA, cell mass,and targeted chemicals, more specifically IPA, 4-HB, or BDO, from: 1)methanol and CO, 2) methanol, CO₂, and H₂, 3) methanol, CO, CO₂, and H₂,4) methanol and synthesis gas comprising CO and H₂, 5) methanol andsynthesis gas comprising CO, CO₂, and H₂, 6) one or more carbohydrates,7) methanol and one or more carbohydrates, and 8) methanol. Exemplarycarbohydrates include but are not limited to glucose, sucrose, xylose,arabinose and glycerol.

Successfully engineering pathways into an organism involves identifyingan appropriate set of enzymes, cloning their corresponding genes into aproduction host, optimizing the stability and expression of these genes,optimizing fermentation conditions, and assaying for product formationfollowing fermentation. A number of enzymes catalyze each step of thepathways for the conversion of synthesis gas and methanol to acetyl-CoA,and further to isopropanol, 4-hydroxybutyrate, or 1,4-butanediol. Toengineer a production host for the utilization of syngas and methanol,one or more exogenous DNA sequence(s) encoding the enzymes can beexpressed in the microorganism.

In some embodiments, the present invention provides a non-naturallyoccurring microbial organism having an isopropanol pathway that includesat least one exogenous nucleic acid encoding an isopropanol pathwayenzyme expressed in a sufficient amount to produce isopropanol. Theisopropanol pathway enzyme includes an acetoacetyl-CoA thiolase, anacetoacetyl-CoA transferase, an acetoacetate decarboxylase, and anisopropanol dehydrogenase. In some embodiments, an isopropanol pathwayof the present invention includes a succinyl-CoA:3-ketoacid-CoAtransferase (SCOT), an acetoacetate decarboxylase, and an isopropanoldehydrogenase. SCOT is a type of acetoacetyl-CoA transferase.

The use of a succinyl-CoA:3-ketoacid-CoA transferase confers a multitudeof benefits when converting acetoacetyl-CoA to acetoacetate as part ofan isopropanol synthesis pathway: 1) Succinyl-CoA:3-ketoacid-CoAtransferase can function as part of a pathway from acetyl-CoA toisopropanol that also generates an energetic equivalent (e.g., ATP,GTP); 2) Succinyl-CoA:3-ketoacid-CoA will not compete with the productpathway for acetyl-CoA substrate; 3) Succinyl-CoA:3-ketoacid-CoA relieson a CoA donor/acceptor molecule, succinate, that is naturallysynthesized in most organisms for industrial production.

The non-naturally occurring organisms of the invention are moreefficient from an energetic standpoint compared to organisms thatutilize acetoacetyl-CoA hydrolase for isopropanol production.Acetoacetyl-CoA hydrolase removes the CoA group from acetoacetyl-CoAwithout the generation of any energetic equivalents.Succinyl-CoA:3-ketoacid-CoA transferase in combination with, forexample, succinyl-CoA synthetase enables the net generation of oneenergetic equivalent (e.g., GTP, ATP). Succinyl-CoA synthetase is anearly ubiquitously present enzyme and is an integral component of thewell-known tricarboxylic acid cycle.

Acetyl-CoA:acetoacetyl-CoA transferase can be used in combination withacetate kinase and phosphotransacetylase or an ADP-forming acetyl-CoAsynthetase to generate an energetic equivalent. Such combinations aremore advantageous from an energetic standpoint than usingacetoacetyl-CoA hydrolase. However, acetate kinase,phosphotransacetylase, and ADP-forming acetyl-CoA synthetase arereversible which can lead to the formation of substantial amounts ofacetate byproduct. Succinyl-CoA synthetase has a lower activity or noactivity at all on acetyl-CoA compared to phosphotransacetylase or anADP-forming acetyl-CoA synthetase.

It is thus desirable in the case of isopropanol to use a CoAdonor/acceptor molecule that is completely disconnected from the productpathway. Butyryl-CoA:acetoacetyl-CoA transferase is one such enzyme andis taught by Subbian et al., US 2008/0293125. However, butyrate is notnaturally present at substantial levels in most organisms including theindustrial workhouse organisms Escherichia coli and Saccharomycescerevisiae. Thus the capability of synthesizing a catalytic amount ofbutyrate would have to engineered into such organisms to enablebutyryl-CoA:acetoacetyl-CoA transferase to function as part of anisopropanol synthesis pathway. Butyrate is also a foul smelling compoundand substantial leakage of butyrate out of the organism could requirespecial process engineering steps to ensure that it is not released intothe air. On the other hand, succinate is non-odorous and is synthesizedin a plethora of organisms including E. coli and S. cerevisiae. Typicalsuccinate forming enzymes include succinyl-CoA synthetase andalpha-ketoglutarate dehydrogenase, both components of the well-knowntricarboxylic acid cycle.

Such organisms may also include at least one enzyme or polypeptide suchas a corrinoid protein, a methyltetrahydrofolate:corrinoid proteinmethyltransferase, a corrinoid iron-sulfur protein, a nickel-proteinassembly protein, a ferredoxin, an acetyl-CoA synthase, a carbonmonoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and ahydrogenase.

In some embodiments, organisms having an isopropanol pathway have amethanol methyltransferase. In such embodiments, the organisms utilize afeedstock such as 1) methanol and CO, 2) methanol, CO₂, and H₂, 3)methanol, CO, CO₂, and H₂, 4) methanol and synthesis gas comprising COand H₂, 5) methanol and synthesis gas comprising CO, CO₂, and H₂, 6) oneor more carbohydrates, 7) methanol and one or more carbohydrates, and 8)methanol. Exemplary carbohydrates include but are not limited toglucose, sucrose, xylose, arabinose and glycerol.

In other embodiments, organisms of the present invention have a formatedehydrogenase, a formyltetrahydrofolate synthetase, amethenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase. Such organismsutilize a feedstock selected from the group consisting of: 1) CO, 2) CO₂and H₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂, 5)synthesis gas comprising CO, CO₂, and H₂, and 6) one or morecarbohydrates. Exemplary carbohydrates include but are not limited toglucose, sucrose, xylose, arabinose, and glycerol.

The invention also provides a non-naturally occurring microbial organismhaving a 4-hydroxybutryate pathway that includes at least one exogenousnucleic acid encoding an 4-hydroxybutryate pathway enzyme expressed in asufficient amount to produce 4-hydroxybutryate. The 4-hydroxybutryatepathway enzyme includes an acetoacetyl-CoA thiolase, a3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoAhydratase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoAhydrolase, a 4-hydroxybutyryl-CoA synthetase, aphosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase.

Such organisms can also include at least one enzyme or polypeptide suchas a corrinoid protein, a methyltetrahydrofolate:corrinoid proteinmethyltransferase, a corrinoid iron-sulfur protein, a nickel-proteinassembly protein, a ferredoxin, an acetyl-CoA synthase, a carbonmonoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and ahydrogenase.

In some embodiments, organisms that have a 4-hydroxybutyrate pathway caninclude a methanol methyltransferase. Such organisms utilize a feedstocksuch as 1) methanol and CO, 2) methanol, CO₂, and H₂, 3) methanol, CO,CO₂, and H₂, 4) methanol and synthesis gas comprising CO and H₂, 5)methanol and synthesis gas comprising CO, CO₂, and H₂, 6) one or morecarbohydrates, 7) methanol and one or more carbohyrates, and 8)methanol. Exemplary carbohydrates include but are not limited toglucose, sucrose, xylose, arabinose and glycerol.

Other organisms that have a 4-hydroxybutyrate pathway can have a formatedehydrogenase, a formyltetrahydrofolate synthetase, amethenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase. Such organismsutilize a feedstock such as 1) CO, 2) CO₂ and H₂, 3) CO and CO₂, 4)synthesis gas comprising CO and H₂, 5) synthesis gas comprising CO, CO₂,and H₂, and 6) one or more carbohydrates. Exemplary carbohydratesinclude but are not limited to glucose, sucrose, xylose, arabinose, andglycerol.

The present invention also provides a non-naturally occurring microbialorganism having a 1,4-butanediol pathway that includes at least oneexogenous nucleic acid encoding a 1,4-butanediol pathway enzymeexpressed in a sufficient amount to produce 1,4-butanediol. The1,4-butanediol pathway enzyme include, for example, an acetoacetyl-CoAthiolase, a 3-Hydroxybutyryl-CoA dehydrogenase, a crotonase, acrotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase (alcoholforming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyrate reductase, aphosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyrate kinase, and a1,4-butanediol dehydrogenase.

Such organisms can also include at least one enzyme or polypeptide suchas a corrinoid protein, a methyltetrahydrofolate:corrinoid proteinmethyltransferase, a corrinoid iron-sulfur protein, a nickel-proteinassembly protein, a ferredoxin, an acetyl-CoA synthase, a carbonmonoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and ahydrogenase.

In some embodiments, an organism having a 1,4-butanediol pathway caninclude a methanol methyltransferase. Such organisms utilize a feedstocksuch as 1) methanol and CO, 2) methanol, CO₂, and H₂, 3) methanol, CO,CO₂, and H₂, 4) methanol and synthesis gas comprising CO and H₂, 5)methanol and synthesis gas comprising CO, CO₂, and H₂, 6) one or morecarbohydrates, 7) methanol and one or more carbohyrates, and 8)methanol. Exemplary carbohydrates include but are not limited toglucose, sucrose, xylose, arabinose and glycerol.

In other embodiments, an organism having a 1,4-butanediol pathway caninclude a formate dehydrogenase, a formyltetrahydrofolate synthetase, amethenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase. Such organismsutilize a feedstock selected from the group consisting of: 1) CO, 2) CO₂and H₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂, 5)synthesis gas comprising CO, CO₂, and H₂, and 6) one or morecarbohydrates. Exemplary carbohydrates include but are not limited toglucose, sucrose, xylose, arabinose, and glycerol.

Also disclosed herein is a non-naturally occurring microbial organismhaving an acetyl-CoA pathway that includes at least one exogenousnucleic acid encoding an acetyl-CoA pathway enzyme expressed in asufficient amount to produce acetyl-CoA. The acetyl-CoA pathway enzymeincludes a methanol methyltransferase and an acetyl-CoA synthase.

In still further embodiments, the present invention provides anon-naturally occurring microbial organism having an isopropanol pathwaycomprising at least one exogenous nucleic acid encoding an isopropanolpathway enzyme expressed in a sufficient amount to produce isopropanol,said isopropanol pathway enzyme comprising a methanol methyltransferase,a corrinoid protein, a methyltetrahydrofolate:corrinoid proteinmethyltransferase, a corrinoid iron-sulfur protein, a nickel-proteinassembly protein, a ferredoxin, an acetyl-CoA synthase, a carbonmonoxide dehydrogenase, a pyruvate ferredoxin oxidoreductase, and ahydrogenase.

In some embodiments, such an organism can include an exogenouspolypeptide or enzyme such as an acetoacetyl-CoA thiolase, anacetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, anacetoacetyl-CoA synthetase, a phosphotransacetoacetylase, anacetoacetate kinase, an acetoacetate decarboxylase, and an isopropanoldehydrogenase. In additional embodiments, such an organism can includean exogenous polypeptide or enzyme such as a succinyl-CoA:3-ketoacid-CoAtransferase (SCOT), an acetoacetate decarboxylase, and an isopropanoldehydrogenase.

Expression of the modified Wood-Ljungdahl pathway in a foreign host (seeFIG. 2B) requires a set of methyltransferases to utilize the carbon andhydrogen provided by methanol and the carbon provided by CO and/or CO₂.A complex of 3 methyltransferase proteins, denoted MtaA, MtaB, and MtaC,perform the desired methanol methyltransferase activity (Naidu andRagsdale, J Bacteriol. 183:3276-3281 (2001); Ragsdale, S. W., Crit Rev.Biochem. Mol. Biol 39:165-195 (2004); Sauer et al., Eur. J Biochem.243:670-677 (1997); Tallant and Krzycki, J. Bacteriol. 178:1295-1301(1996); Tallant and Krzycki, J Bacteriol. 179:6902-6911 (1997); Tallantet al., J Biol Chem. 276:4485-4493 (2001)).

MtaB is a zinc protein that catalyzes the transfer of a methyl groupfrom methanol to MtaC, a corrinoid protein. Exemplary genes encodingMtaB and MtaC can be found in methanogenic archaea such asMethanosarcina barkeri (Maeder et al., J Bacteriol. 188:7922-7931(2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res12:532-542 (2002)), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007)). In general, the MtaB and MtaC genesare adjacent to one another on the chromosome as their activities aretightly interdependent. The protein sequences of various MtaB and MtaCencoding genes in M. barkeri, M. acetivorans, and M. thermoaceticum canbe identified by their following GenBank accession numbers.

TABLE 1 Protein GenBank ID GI Number Organism MtaB1 YP_304299 73668284Methanosarcina barkeri MtaC1 YP_304298 73668283 Methanosarcina barkeriMtaB2 YP_307082 73671067 Methanosarcina barkeri MtaC2 YP_307081 73671066Methanosarcina barkeri MtaB3 YP_304612 73668597 Methanosarcina barkeriMtaC3 YP_304611 73668596 Methanosarcina barkeri MtaB1 NP_615421 20089346Methanosarcina acetivorans MtaB1 NP_615422 20089347 Methanosarcinaacetivorans MtaB2 NP_619254 20093179 Methanosarcina acetivorans MtaC2NP_619253 20093178 Methanosarcina acetivorans MtaB3 NP_616549 20090474Methanosarcina acetivorans MtaC3 NP_616550 20090475 Methanosarcinaacetivorans MtaB YP_430066 83590057 Moorella thermoacetica MtaCYP_430065 83590056 Moorella thermoacetica

The MtaB1 and MtaC1 genes, YP_(—)304299 and YP_(—)304298, from M.barkeri were cloned into E. coli and sequenced (Sauer et al., Eur. JBiochem. 243:670-677 (1997)). The crystal structure of thismethanol-cobalamin methyltransferase complex is also available(Hagemeier et al., Proc Natl Acad Sci U S.A 103:18917-18922 (2006)). TheMtaB genes, YP_(—)307082 and YP_(—)304612, in M. barkeri were identifiedby sequence homology to YP_(—)304299. In general, homology searches arean effective means of identifying methanol methyltransferases becauseMtaB encoding genes show little or no similarity to methyltransferasesthat act on alternative substrates such as trimethylamine,dimethylamine, monomethylamine, or dimethylsulfide. The MtaC genes,YP_(—)307081 and YP_(—)304611, were identified based on their proximityto the MtaB genes and also their homology to YP_(—)304298. The threesets of MtaB and MtaC genes from M. acetivorans have been genetically,physiologically, and biochemically characterized (Pritchett and Metcalf,Mol. Microbiol 56:1183-1194 (2005)). Mutant strains lacking two of thesets were able to grow on methanol, whereas a strain lacking all threesets of MtaB and MtaC genes sets could not grow on methanol. Thisindicates that each set of genes plays a role in methanol utilization.The M. thermoacetica MtaB gene was identified based on homology to themethanogenic MtaB genes and also by its adjacent chromosomal proximityto the methanol-induced corrinoid protein, MtaC, which has beencrystallized (Zhou et al., Acta Crystallogr. Sect. F Struct. Biol Cryst.Commun. 61:537-540 (2005)) and further characterized by Northernhybridization and Western Blotting (Das et al., Proteins 67:167-176(2007)).

MtaA is zinc protein that catalyzes the transfer of the methyl groupfrom MtaC to either Coenzyme M in methanogens or methyltetrahydrofolatein acetogens. MtaA can also utilize methylcobalamin as the methyl donor.Exemplary genes encoding MtaA can be found in methanogenic archaea suchas Methanosarcina barkeri (Maeder et al., J Bacteriol. 188:7922-7931(2006)) and Methanosarcina acetivorans (Galagan et al., Genome Res12:532-542 (2002)), as well as the acetogen, Moorella thermoacetica (Daset al., Proteins 67:167-176 (2007)). In general, MtaA proteins thatcatalyze the transfer of the methyl group from CH₃-MtaC are difficult toidentify bioinformatically as they share similarity to other corrinoidprotein methyltransferases and are not oriented adjacent to the MtaB andMtaC genes on the chromosomes. Nevertheless, a number of MtaA encodinggenes have been characterized. The protein sequences of these genes inM. barkeri and M. acetivorans can be identified by the following GenBankaccession numbers.

TABLE 2 Protein GenBank ID GI Number Organism MtaA YP_304602 73668587Methanosarcina barkeri MtaA1 NP_619241 20093166 Methanosarcinaacetivorans MtaA2 NP_616548 20090473 Methanosarcina acetivorans

The MtaA gene, YP_(—)304602, from M. barkeri was cloned, sequenced, andfunctionally overexpressed in E. coli (Harms and Thauer, Eur. J Biochem.235:653-659 (1996)). In M. acetivorans, MtaA1 is required for growth onmethanol, whereas MtaA2 is dispensable even though methane productionfrom methanol is reduced in MtaA2 mutants (Bose et al., J Bacteriol.190:4017-4026 (2008)). There are multiple additional MtaA homologs in M.barkeri and M. acetivorans that are as yet uncharacterized, but can alsocatalyze corrinoid protein methyltransferase activity.

Putative MtaA encoding genes in M. thermoacetica were identified bytheir sequence similarity to the characterized methanogenic MtaA genes.Specifically, three M. thermoacetica genes show high homology (>30%sequence identity) to YP_(—)304602 from M. barkeri. Unlike methanogenicMtaA proteins that naturally catalyze the transfer of the methyl groupfrom CH₃-MtaC to Coenzyme M, an M. thermoacetica MtaA is likely totransfer the methyl group to methyltetrahydrofolate given the similarroles of methyltetrahydrofolate and Coenzyme M in methanogens andacetogens, respectively. The protein sequences of putative MtaA encodinggenes from M. thermoacetica can be identified by the following GenBankaccession numbers.

TABLE 3 Protein GenBank ID GI Number Organism MtaA YP_430937 83590928Moorella thermoacetica MtaA YP_431175 83591166 Moorella thermoaceticaMtaA YP_430935 83590926 Moorella thermoacetica

ACS/CODH is the central enzyme of the carbonyl branch of theWood-Ljungdahl pathway. It catalyzes the reversible reduction of carbondioxide to carbon monoxide and also the synthesis of acetyl-CoA fromcarbon monoxide, Coenzyme A, and the methyl group from a methylatedcorrinoid-iron-sulfur protein. The corrinoide-iron-sulfur-protein ismethylated by methyltetrahydrofolate via a methyltransferase. Expressionof ACS/CODH in a foreign host can be done by introducing one or more ofthe following proteins and their corresponding activities:Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE),Corrinoid iron-sulfur protein (AcsD), Nickel-protein assembly protein(AcsF), Ferredoxin (Orf7), Acetyl-CoA synthase (AcsB and AcsC), Carbonmonoxide dehydrogenase (AcsA) or Nickel-protein assembly protein (CooC).

The genes used for carbon-monoxide dehydrogenase/acetyl-CoA synthaseactivity typically reside in a limited region of the native genome thatmay be an extended operon (Morton et al., J Biol Chem. 266:23824-23828(1991); Ragsdale, S. W., Crit Rev. Biochem. Mol. Biol 39:165-195 (2004);Roberts et al., Proc Natl Acad Sci U S.A 86:32-36 (1989)). Each of thegenes in this operon from the acetogen, M. thermoacetica, has alreadybeen cloned and expressed actively in E. coli (Lu et al., J Biol Chem.268:5605-5614 (1993); Roberts et al., Proc Natl Acad Sci U S.A 86:32-36(1989)). The protein sequences of these genes can be identified by thefollowing GenBank accession numbers.

TABLE 4 Protein GenBank ID GI Number Organism AcsE YP_430054 83590045Moorella thermoacetica AcsD YP_430055 83590046 Moorella thermoaceticaAcsF YP_430056 83590047 Moorella thermoacetica Orf7 YP_430057 83590048Moorella thermoacetica AcsC YP_430058 83590049 Moorella thermoaceticaAcsB YP_430059 83590050 Moorella thermoacetica AcsA YP_430060 83590051Moorella thermoacetica CooC YP_430061 83590052 Moorella thermoacetica

The hydrogenogenic bacterium, Carboxydothermus hydrogenoformans, canutilize carbon monoxide as a growth substrate by means of acetyl-CoAsynthase (Wu et al., PLoS Genet. 1:e65 (1995)). In strain Z-2901, theacetyl-CoA synthase enzyme complex lacks carbon monoxide dehydrogenasedue to a frameshift mutation (Wu et al., PLoS Genet. 1:e65 (1995)),whereas in strain DSM 6008, a functional unframeshifted full-lengthversion of this protein has been purified (Svetlitchnyi et al., ProcNatl Acad Sci U S.A 101:446-451 (2004)). The protein sequences of the C.hydrogenoformans genes from strain Z-2901 are identified by thefollowing GenBank accession numbers. Sequences for Carboxydothermushydrogenoformans DSM 6008 are not yet accessible in publicly availabledatabases.

TABLE 5 Protein GenBank ID GI Number Organism AcsE YP_360065 78044202Carboxydothermus hydrogenoformans AcsD YP_360064 78042962Carboxydothermus hydrogenoformans AcsF YP_360063 78044060Carboxydothermus hydrogenoformans Orf7 YP_360062 78044449Carboxydothermus hydrogenoformans AcsC YP_360061 78043584Carboxydothermus hydrogenoformans AcsB YP_360060 78042742Carboxydothermus hydrogenoformans CooC YP_360059 78044249Carboxydothermus hydrogenoformans

Homologous ACS/CODH genes can also be found in the draft genome assemblyof Clostridium carboxidivorans P7.

TABLE 6 Protein GenBank ID GI Number Organism AcsA ZP_05392944.1255526020 Clostridium carboxidivorans P7 CooC ZP_05392945.1 255526021Clostridium carboxidivorans P7 AcsF ZP_05392952.1 255526028 Clostridiumcarboxidivorans P7 AcsD ZP_05392953.1 255526029 Clostridiumcarboxidivorans P7 AcsC ZP_05392954.1 255526030 Clostridiumcarboxidivorans P7 AcsE ZP_05392955.1 255526031 Clostridiumcarboxidivorans P7 AcsB ZP_05392956.1 255526032 Clostridiumcarboxidivorans P7 Orf7 ZP_05392958.1 255526034 Clostridiumcarboxidivorans P7

The methanogenic archaeon, Methanosarcina acetivorans, can also grow oncarbon monoxide, exhibits acetyl-CoA synthase/carbon monoxidedehydrogenase activity, and produces both acetate and formate (Lessneret al., Proc Natl Acad Sci U S.A 103:17921-17926 (2006)). This organismcontains two sets of genes that encode ACS/CODH activity (Rother andMetcalf Proc Natl Acad Sci U S.A 101:16929-16934 (2004)). The proteinsequences of both sets of M. acetivorans genes can be identified by thefollowing GenBank accession numbers.

TABLE 7 Protein GenBank ID GI Number Organism AcsC NP_618736 20092661Methanosarcina acetivorans AcsD NP_618735 20092660 Methanosarcinaacetivorans AcsF, CooC NP_618734 20092659 Methanosarcina acetivoransAcsB NP_618733 20092658 Methanosarcina acetivorans AcsEps NP_61873220092657 Methanosarcina acetivorans AcsA NP_618731 20092656Methanosarcina acetivorans AcsC NP_615961 20089886 Methanosarcinaacetivorans AcsD NP_615962 20089887 Methanosarcina acetivorans AcsF,CooC NP_615963 20089888 Methanosarcina acetivorans AcsB NP_61596420089889 Methanosarcina acetivorans AcsEps NP_615965 20089890Methanosarcina acetivorans AcsA NP_615966 20089891 Methanosarcinaacetivorans

The AcsC, AcsD, AcsB, AcsEps, and AcsA proteins are commonly referred toas the gamma, delta, beta, epsilon, and alpha subunits of themethanogenic CODH/ACS. Homologs to the epsilon encoding genes are notpresent in acetogens such as M. thermoacetica or hydrogenogenic bacteriasuch as C. hydrogenoformans. Hypotheses for the existence of two activeCODH/ACS operons in M. acetivorans include catalytic properties (i.e.,K_(m), V_(max), k_(cat)) that favor carboxidotrophic or aceticlasticgrowth or differential gene regulation enabling various stimuli toinduce CODH/ACS expression (Rother et al., Arch. Microbiol 188:463-472(2007)).

In M. thermoacetica, C. hydrogenoformans and C. carboxidivorans P7,additional CODH encoding genes are located outside of the ACS/CODHoperons. These enzymes provide a means for extracting electrons (orreducing equivalents) from the conversion of carbon monoxide to carbondioxide. The reducing equivalents are then passed to accepters such asoxidized ferredoxin, NADP+, water, or hydrogen peroxide to form reducedferredoxin, NADPH, H₂, or water, respectively. In some cases,hydrogenase encoding genes are located adjacent to a CODH. InRhodospirillum rubrum, the encoded CODH/hydrogenase proteins form amembrane-bound enzyme complex that is proposed to be a site whereenergy, in the form of a proton gradient, is generated from theconversion of CO to CO₂ and H₂ (Fox et al., J Bacteriol. 178:6200-6208(1996)). The CODH-I of C. hydrogenoformans and its adjacent genes havebeen proposed to catalyze a similar functional role based on theirsimilarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al.,PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-I was also shownto exhibit intense CO oxidation and CO₂ reduction activities when linkedto an electrode (Parkin et al., J Am. Chem. Soc. 129:10328-10329(2007)). The genes encoding the C. hydrogenoformans CODH-II and CooF, aneighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMSMicrobiol Lett. 191:243-247 (2000)). The resulting complex wasmembrane-bound, although cytoplasmic fractions of CODH-II were shown tocatalyze the formation of NADPH suggesting an anabolic role(Svetlitchnyi et al., J Bacteriol. 183:5134-5144 (2001)). The crystalstructure of the CODH-II is also available (Dobbek et al., Science293:1281-1285 (2001)). The protein sequences of exemplary CODH andhydrogenase genes can be identified by the following GenBank accessionnumbers.

TABLE 8 Protein GenBank ID GI Number Organism CODH (putative) YP_43081383590804 Moorella thermoacetica CODH-I (CooS-I) YP_360644 78043418Carboxydothermus hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooM AAC45116 1515466 Rhodospirillumrubrum CooK AAC45117 1515467 Rhodospirillum rubrum CooL AAC45118 1515468Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooUAAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH(CooS) AAC45123 1498748 Rhodospirillum rubrum CooC AAC45124 1498749Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJAAC45126 1498751 Rhodospirillum rubrum CODH-II (CooS-II) YP_35895778044574 Carboxydothermus hydrogenoformans CooF YP_358958 78045112Carboxydothermus hydrogenoformans CODH (putative) ZP_05390164.1255523193 Clostridium carboxidivorans P7 ZP_05390341.1 255523371Clostridium carboxidivorans P7 ZP_05391756.1 255524806 Clostridiumcarboxidivorans P7 ZP_05392944.1 255526020 Clostridium carboxidivoransP7 CooF ZP_05394386.1 255527518 Clostridium carboxidivorans P7 CooFZP_05394384.1 255527516 Clostridium carboxidivorans P7 HypAZP_05392031.1 255525086 Clostridium carboxidivorans P7 CooHZP_05392429.1 255525492 Clostridium carboxidivorans P7 CooLZP_05392428.1 255525491 Clostridium carboxidivorans P7 CooMZP_05392434.1 255525497 Clostridium carboxidivorans P7

Anaerobic growth on synthesis gas and methanol in the absence of anexternal electron acceptor is conferred upon the host organism with MTRand ACS/CODH activity by providing pyruvate synthesis via pyruvateferredoxin oxidoreductase (PFOR). The PFOR from Desulfovibrio africanushas been cloned and expressed in E. coli resulting in an activerecombinant enzyme that was stable for several days in the presence ofoxygen (Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygenstability is relatively uncommon in PFORs and is reported to beconferred by a 60 residue extension in the polypeptide chain of the D.africanus enzyme. The M. thermoacetica PFOR is also well characterized(Menon and Ragsdale, Biochemistry 36:8484-8494 (1997)) and was shown tohave high activity in the direction of pyruvate synthesis duringautotrophic growth (Furdui and Ragsdale, J Biol Chem. 275:28494-28499(2000)). Further, E. coli possesses an uncharacterized open readingframe, ydbK, that encodes a protein that is 51% identical to the M.thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E.coli has been described (Blaschkowski et al., Eur. J Biochem.123:563-569 (1982)). Homologs also exist in C. carboxidivorans P7. Theprotein sequences of these exemplary PFOR enzymes are identified by thefollowing GenBank accession numbers shown in Table 9 below. Severaladditional PFOR enzymes are described in Ragsdale, S. W., Chem. Rev.103:2333-2346 (2003). Finally, flavodoxin reductases (e.g., fqrB fromHelicobacter pylori or Campylobacter jejuni) (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al.,Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J.Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPHfrom the reduced ferredoxin generated by PFOR. These proteins areidentified below in Table 9.

TABLE 9 Protein GenBank ID GI Number Organism Por CAA70873.1 1770208Desulfovibrio africanus Por YP_428946.1 83588937 Moorella thermoaceticaYdbK NP_415896.1 16129339 Escherichia coli Por ZP_05392150.1 255525208Clostridium carboxidivorans P7 Por ZP_05390930.1 255523967 Clostridiumcarboxidivorans P7 Por ZP_05394396.1 255527529 Clostridiumcarboxidivorans P7 Por ZP_05394397.1 255527530 Clostridiumcarboxidivorans P7 FqrB NP_207955.1 15645778 Helicobacter pylori FqrBYP_001482096.1 157414840 Campylobacter jejuni RnfC EDK33306.1 146346770Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfGEDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1 146346773Clostridium kluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfBEDK33311.1 146346775 Clostridium kluyveri

The conversion of pyruvate into acetyl-CoA can be catalyzed by severalother enzymes or their combinations thereof. For example, pyruvatedehydrogenase can transform pyruvate into acetyl-CoA with theconcomitant reduction of a molecule of NAD into NADH. It is amulti-enzyme complex that catalyzes a series of partial reactions whichresults in acylating oxidative decarboxylation of pyruvate. The enzymecomprises of three subunits: the pyruvate decarboxylase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase(E3). This enzyme is naturally present in several organisms, includingE. coli and S. cerevisiae. In the E. coli enzyme, specific residues inthe E1 component are responsible for substrate specificity (Bisswanger,H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem.8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653 (2000)).Enzyme engineering efforts have improved the E. coli PDH enzyme activityunder anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858(2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhouet al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coliPDH, the B. subtilis complex is active and required for growth underanaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755(1997)). The Klebsiella pneumoniae PDH, characterized during growth onglycerol, is also active under anaerobic conditions (Menzel et al., J.Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme complexfrom bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U.S.A.98:14802-14807 (2001)) and the E2 catalytic domain from Azotobactervinelandii are available (Mattevi et al., Science 255:1544-1550 (1992)).Yet another enzyme that can catalyze this conversion is pyruvate formatelyase. This enzyme catalyzes the conversion of pyruvate and CoA intoacetyl-CoA and formate. Pyruvate formate lyase is a common enzyme inprokaryotic organisms that is used to help modulate anaerobic redoxbalance. Exemplary enzymes can be found in Escherichia coli encoded bypflB (Knappe and Sawers, FEMS. Microbiol Rev. 6:383-398 (1990)),Lactococcus lactis (Melchiorsen et al., Appl. Microbiol. Biotechnol.58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al.,Oral. Microbiol Immunol. 18:293-297 (2003)). E. coli possesses anadditional pyruvate formate lyase, encoded by tdcE, that catalyzes theconversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA,respectively (Hesslinger et al., Mol. Microbiol. 27:477-492 (1998)).Both pflB and tdcE from E. coli require the presence of pyruvate formatelyase activating enzyme, encoded by pflA. Further, a short proteinencoded by yfiD in E. coli can associate with and restore activity tooxygen-cleaved pyruvate formate lyase (Vey et al., Proc. Natl. Acad.Sci. U.S.A. 105:16137-16141 (2008). Note that pflA and pflB from E. coliwere expressed in S. cerevisiae as a means to increase cytosolicacetyl-CoA for butanol production as described in WO/2008/080124].Additional pyruvate formate lyase and activating enzyme candidates,encoded by pfl and act, respectively, are found in Clostridiumpasteurianum (Weidner et al., J Bacteriol. 178:2440-2444 (1996)).

Further, different enzymes can be used in combination to convertpyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA isobtained in the cytosol by first decarboxylating pyruvate to formacetaldehyde; the latter is oxidized to acetate by acetaldehydedehydrogenase and subsequently activated to form acetyl-CoA byacetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme inseveral other organisms including E. coli (Kumari et al., J. Bacteriol.177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology151:3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205(2005)), and Moorella thermoacetica (described already). Alternatively,acetate can be activated to form acetyl-CoA by acetate kinase andphosphotransacetylase. Acetate kinase first converts acetate intoacetyl-phosphate with the accompanying use of an ATP molecule.Acetyl-phosphate and CoA are next converted into acetyl-CoA with therelease of one phosphate by phosphotransacetylase. Both acetate kinaseand phosphotransacetylyase are well-studied enzymes in severalClostridia and Methanosarcina thermophila.

Yet another way of converting pyruvate to acetyl-CoA is via pyruvateoxidase. Pyruvate oxidase converts pyruvate into acetate, usingubiquione as the electron acceptor. In E. coli, this activity is encodedby poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiaeand Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor(Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al.,Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem.255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor.Acetate can then be converted into acetyl-CoA by either acetyl-CoAsynthetase or by acetate kinase and phosphotransacetylase, as describedearlier. Some of these enzymes can also catalyze the reverse reactionfrom acetyl-CoA to pyruvate.

Unlike the redox neutral conversion of CO and MeOH to acetyl-CoA oracetate, the production of more highly reduced products such as ethanol,butanol, isobutanol, isopropanol, 1,4-butanediol, succinic acid, fumaricacid, malic acid, 4-hydroxybutyric acid, 3-hydroxypropionic acid, lacticacid, adipic acid, 6-aminocaproic acid, hexamethylenediamine,3-hydroxyisobutyric acid, 2-hydroxyisobutyric acid, methacrylic acid,and acrylic acid at the highest possible yield requires the extractionof additional reducing equivalents from both CO and H₂ (for example, seeethanol formation in FIG. 2A). Specifically, reducing equivalents (e.g.,2 [H] in FIG. 2) are obtained by the conversion of CO and water to CO₂via carbon monoxide dehydrogenase or directly from the activity of ahydrogen-utilizing hydrogenase which transfers electrons from H₂ to anacceptor such as ferredoxin, flavodoxin, FAD⁺, NAD⁺, or NADP⁺.

Native to E. coli and other enteric bacteria are multiple genes encodingup to four hydrogenases (Sawers, G., Antonie Van Leeuwenhoek 66:57-88(1994); Sawers et al., J Bacteriol. 164:1324-1331 (1985); Sawers andBoxer, Eur. J Biochem. 156:265-275 (1986); Sawers et al., J Bacteriol.168:398-404 (1986)). Given the multiplicity of enzyme activities E. colior another host organism can provide sufficient hydrogenase activity tosplit incoming molecular hydrogen and reduce the corresponding acceptor.Among the endogenous hydrogen-lyase enzymes of E. coli are hydrogenase3, a membrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyf gene clusters, respectively. Hydrogenaseactivity in E. coli is also dependent upon the expression of the hypgenes whose corresponding proteins are involved in the assembly of thehydrogenase complexes (Jacobi et al., Arch. Microbiol 158:444-451(1992); Rangarajan et al., J Bacteriol. 190:1447-1458 (2008)). The M.thermoacetica hydrogenases are suitable for a host that lacks sufficientendogenous hydrogenase activity. M. thermoacetica can grow with CO₂ asthe exclusive carbon source indicating that reducing equivalents areextracted from H₂ to enable acetyl-CoA synthesis via the Wood-Ljungdahlpathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake andDaniel, Res Microbiol 155:869-883 (2004); Kellum and Drake, J Bacteriol.160:466-469 (1984)) (see FIG. 2A). M. thermoacetica has homologs toseveral hyp, hyc, and hyf genes from E. coli. These protein sequencesencoded for by these genes are identified by the following GenBankaccession numbers. In addition, several gene clusters encodinghydrogenase functionality are present in M. thermoacetica and theircorresponding protein sequences are also provided below in Table 10.

TABLE 10 Protein GenBank ID GI Number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyp genes are shown below in Table 11. Hydrogenase 3 proteins are listedin Table 12. Hydrogenase 4 proteins are shown in Table 13.

TABLE 11 Protein GenBank ID GI Number Organism Moth_2175 YP_43100783590998 Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoacetica

TABLE 12 Protein GenBank ID GI Number Organism HycA NP_417205 16130632Escherichia coli HycB NP_417204 16130631 Escherichia coli HycC NP_41720316130630 Escherichia coli HycD NP_417202 16130629 Escherichia coli HycENP_417201 16130628 Escherichia coli HycF NP_417200 16130627 Escherichiacoli HycG NP_417199 16130626 Escherichia coli HycH NP_417198 16130625Escherichia coli HycI NP_417197 16130624 Escherichia coli

TABLE 13 Protein GenBank ID GI Number Organism HyfA NP_416976 90111444Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC NP_41697890111445 Escherichia coli HyfD NP_416979 16130409 Escherichia coli HyfENP_416980 16130410 Escherichia coli HyfF NP_416981 16130411 Escherichiacoli HyfG NP_416982 16130412 Escherichia coli HyfH NP_416983 16130413Escherichia coli HyfI NP_416984 16130414 Escherichia coli HyfJ NP_41698590111446 Escherichia coli HyfR NP_416986 90111447 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. colihyc and/or hyf genes are shown in Table 14 below. Additionalhydrogenase-encoding gene clusters in M. thermoacetica are shown inTable 15.

TABLE 14 Protein GenBank ID GI Number Organism Moth_2182 YP_43101483591005 Moorella thermoacetica Moth_2183 YP_431015 83591006 Moorellathermoacetica Moth_2184 YP_431016 83591007 Moorella thermoaceticaMoth_2185 YP_431017 83591008 Moorella thermoacetica Moth_2186 YP_43101883591009 Moorella thermoacetica Moth_2187 YP_431019 83591010 Moorellathermoacetica Moth_2188 YP_431020 83591011 Moorella thermoaceticaMoth_2189 YP_431021 83591012 Moorella thermoacetica Moth_2190 YP_43102283591013 Moorella thermoacetica Moth_2191 YP_431023 83591014 Moorellathermoacetica Moth_2192 YP_431024 83591015 Moorella thermoacetica

TABLE 15 Protein GenBank ID GI Number Organism Moth_0439 YP_42931383589304 Moorella thermoacetica Moth_0440 YP_429314 83589305 Moorellathermoacetica Moth_0441 YP_429315 83589306 Moorella thermoaceticaMoth_0442 YP_429316 83589307 Moorella thermoacetica Moth_0809 YP_42967083589661 Moorella thermoacetica Moth_0810 YP_429671 83589662 Moorellathermoacetica Moth_0811 YP_429672 83589663 Moorella thermoaceticaMoth_0812 YP_429673 83589664 Moorella thermoacetica Moth_0814 YP_42967483589665 Moorella thermoacetica Moth_0815 YP_429675 83589666 Moorellathermoacetica Moth_0816 YP_429676 83589667 Moorella thermoaceticaMoth_1193 YP_430050 83590041 Moorella thermoacetica Moth_1194 YP_43005183590042 Moorella thermoacetica Moth_1195 YP_430052 83590043 Moorellathermoacetica Moth_1196 YP_430053 83590044 Moorella thermoaceticaMoth_1717 YP_430562 83590553 Moorella thermoacetica Moth_1718 YP_43056383590554 Moorella thermoacetica Moth_1719 YP_430564 83590555 Moorellathermoacetica Moth_1883 YP_430726 83590717 Moorella thermoaceticaMoth_1884 YP_430727 83590718 Moorella thermoacetica Moth_1885 YP_43072883590719 Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorellathermoacetica Moth_1887 YP_430730 83590721 Moorella thermoaceticaMoth_1888 YP_430731 83590722 Moorella thermoacetica Moth_1452 YP_43030583590296 Moorella thermoacetica Moth_1453 YP_430306 83590297 Moorellathermoacetica Moth_1454 YP_430307 83590298 Moorella thermoacetica

Isopropanol production is achieved in recombinant E. coli followingexpression of two heterologous genes from C. acetobutylicum (thl and adcencoding acetoacetyl-CoA thiolase and acetoacetate decarboxylase,respectively) and one from C. beijerinckii (adh encoding a secondaryalcohol dehydrogenase), along with the increased expression of thenative atoA and atoD genes which encode acetoacetyl-CoA:acetate:CoAtransferase activity (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007)).

Acetoacetyl-CoA thiolase converts two molecules of acetyl-CoA into onemolecule each of acetoacetyl-CoA and CoA. Such a conversion is the firststep for isopropanol, 4-hydroxybutyrate, and 1,4-butanediol synthesisfrom acetyl-CoA, as shown in FIGS. 2 and 3. Exemplary acetoacetyl-CoAthiolase enzymes include the gene products of atoB from E. coli (Martinet al., Nat. Biotechnol 21:796-802 (2003)), thlA and thlB from C.acetobutylicum (Hanai et al., Appl Environ Microbiol 73:7814-7818(2007); Winzer et al., J. Mol. Microbiol Biotechnol 2:531-541 (2000),and ERG10 from S. cerevisiae Hiser et al., J. Biol. Chem.269:31383-31389 (1994)).

TABLE 16 Protein GenBank ID GI Number Organism AtoB NP_416728 16130161Escherichia coli ThlA NP_349476.1 15896127 Clostridium acetobutylicumThlB NP_149242.1 15004782 Clostridium acetobutylicum ERG10 NP_0152976325229 Saccharomyces cerevisiae

The conversion of acetoacetyl-CoA to acetoacetate or of4-hydroxybutyryl-CoA to 4-hydroxybutyrate can be carried out by anacetoacetyl-CoA transferase or 4-hydroxybutyryl-CoA transferase,respectively. These enzymes conserve the energy stored in the CoA-esterbonds of acetoacetyl-CoA and 4-hydroxybutyryl-CoA. Many transferaseshave broad specificity and thus can utilize CoA acceptors as diverse asacetate, succinate, propionate, butyrate, 2-methylacetoacetate,3-ketohexanoate, 3-ketopentanoate, valerate, crotonate,3-mercaptopropionate, propionate, vinylacetate, butyrate, among others.Acetoacetyl-CoA transferase catalyzes the conversion of acetoacetyl-CoAto acetoacetate while transferring the CoA moiety to a CoA acceptormolecule. Several exemplary transferase enzymes capable of catalyzingthis transformation are provided below. These enzymes either naturallyexhibit the desired acetoacetyl-CoA transferase activity or they can beengineered via directed evolution to accept acetoacetyl-CoA as asubstrate with increased efficiency. Such enzymes, either naturally orfollowing directed evolution, are also suitable for catalyzing theconversion of 4-hydroxybutyryl-CoA to 4-hydroxybutyrate via atransferase mechanism.

In one embodiment an exemplary acetoacetyl-CoA transferase isacetoacetyl-CoA:acetate-CoA transferase. This enzyme naturally convertsacetate to acetyl-CoA while converting acetoacetyl-CoA to acetoacetate.In another embodiment, a succinyl-CoA:3-ketoacid CoA transferase (SCOT)catalyzes the conversion of the 3-ketoacyl-CoA, acetoacetyl-CoA, to the3-ketoacid, acetoacetate.

Acetoacetyl-CoA:acetyl-CoA transferase naturally convertsacetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA. This enzymecan also accept 3-hydroxybutyryl-CoA as a substrate or could beengineered to do so. Exemplary enzymes include the gene products ofatoAD from E. coli (Hanai et al., Appl Environ Microbiol. 73:7814-7818(2007)), ctfAB from C. acetobutylicum (Jojima et al., Appl MicrobiolBiotechnol. 77:1219-1224 (2008)), and ctfAB from Clostridiumsaccharoperbutylacetonicum (Kosaka et al., Biosci. Biotechnol Biochem.71:58-68 (2007)). Information related to these proteins and genes isshown below:

TABLE 17 Protein GENBANK ID GI NUMBER ORGANISM AtoA P76459.1 2492994Escherichia coli AtoD P76458.1 2492990 Escherichia coli CtfA NP_149326.115004866 Clostridium acetobutylicum CtfB NP_149327.1 15004867Clostridium acetobutylicum CtfA AAP42564.1 31075384 Clostridiumsaccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridiumsaccharoperbutylacetonicum

Succinyl-CoA:3-ketoacid-CoA transferase naturally converts succinate tosuccinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid.Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present inHelicobacter pylori (Corthesy-Theulaz et al., J. Biol. Chem.272:25659-25667 (1997)), Bacillus subtilis (Stols et al., Protein. Expr.Purif. 53:396-403 (2007)), and Homo sapiens (Fukao et al., Genomics68:144-151 (2000); Tanaka et al., Mol. Hum. Reprod. 8:16-23 (2002)).Information related to these proteins and genes is shown below:

TABLE 18 Protein GENBANK ID GI NUMBER ORGANISM HPAG1_0676 YP_627417108563101 Helicobacter pylori HPAG1_0677 YP_627418 108563102Helicobacter pylori ScoA NP_391778 16080950 Bacillus subtilis ScoBNP_391777 16080949 Bacillus subtilis OXCT1 NP_000427 4557817 Homosapiens OXCT2 NP_071403 11545841 Homo sapiens

Additional suitable acetoacetyl-CoA and 4-hydroxybutyryl-CoAtransferases are encoded by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri. These enzymes have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferaseactivity, respectively (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A.105:2128-2133 (2008); Sohling and Gottschalk, J Bacteriol. 178:871-880(1996)). Similar CoA transferase activities are also present inTrichomonas vaginalis (van Grinsven et al., J. Biol. Chem. 283:1411-1418(2008)) and Trypanosoma brucei (Riviere et al., J. Biol. Chem.279:45337-45346 (2004)). Yet another transferase capable of the desiredconversions is butyryl-CoA:acetoacetate CoA-transferase. Exemplaryenzymes can be found in Fusobacterium nucleatum (Barker et al., J.Bacteriol. 152(1):201-7 (1982)), Clostridium SB4 (Barker et al., J.Biol. Chem. 253(4):1219-25 (1978)), and Clostridium acetobutylicum(Wiesenborn et al., Appl. Environ. Microbiol. 55(2):323-9 (1989)).Although specific gene sequences were not provided forbutyryl-CoA:acetoacetate CoA-transferase in these references, the genesFN0272 and FN0273 have been annotated as a butyrate-acetoacetateCoA-transferase (Kapatral et al., J. Bact. 184(7) 2005-2018 (2002)).Homologs in Fusobacterium nucleatum such as FN1857 and FN1856 alsolikely have the desired acetoacetyl-CoA transferase activity. FN1857 andFN1856 are located adjacent to many other genes involved in lysinefermentation and are thus very likely to encode an acetoacetate:butyrateCoA transferase (Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197(2007)). Additional candidates from Porphyrmonas gingivalis andThermoanaerobacter tengcongensis can be identified in a similar fashion(Kreimeyer, et al., J. Biol. Chem. 282 (10) 7191-7197 (2007)).Information related to these proteins and genes is shown below:

TABLE 19 Protein GENBANK ID GI NUMBER ORGANISM Cat1 P38946.1 729048Clostridium kluyveri Cat2 P38942.2 1705614 Clostridium kluyveri Cat3EDK35586.1 146349050 Clostridium kluyveri TVAG_395550 XP_001330176123975034 Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875Trypanosoma brucei FN0272 NP_603179.1 19703617 Fusobacterium nucleatumFN0273 NP_603180.1 19703618 Fusobacterium nucleatum FN1857 NP_602657.119705162 Fusobacterium nucleatum FN1856 NP_602656.1 19705161Fusobacterium nucleatum PG1066 NP_905281.1 34540802 Porphyromonasgingivalis W83 PG1075 NP_905290.1 34540811 Porphyromonas gingivalis W83TTE0720 NP_622378.1 20807207 Thermoanaerobacter tengcongensis MB4TTE0721 NP_622379.1 20807208 Thermoanaerobacter tengcongensis MB4

Acetoacetyl-CoA can be hydrolyzed to acetoacetate by acetoacetyl-CoAhydrolase. Similarly, 4-hydroxybutyryl-CoA can be hydrolyzed to4-hydroxybutyate by 4-hydroxybutyryl-CoA hydrolase. Many CoA hydrolases(EC 3.1.2.1) have broad substrate specificity and are suitable enzymesfor these transformations either naturally or following enzymeengineering. Though the sequences were not reported, severalacetoacetyl-CoA hydrolases were identified in the cytosol andmitochondrion of the rat liver (Aragon and Lowenstein, J. Biol. Chem.258(8):4725-4733 (1983)). Additionally, an enzyme from Rattus norvegicusbrain (Robinson et al., Biochem. Biophys. Res. Commun. 71:959-965(1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. Theacot12 enzyme from the rat liver was shown to hydrolyze C2 to C6acyl-CoA molecules (Suematsu et al., Eur. J. Biochem. 268:2700-2709(2001)). Though its sequence has not been reported, the enzyme from themitochondrion of the pea leaf showed activity on acetyl-CoA,propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, andcrotonyl-CoA (Zeiher and Randall, Plant. Physiol. 94:20-27 (1990)).Additionally, a glutaconate CoA-transferase from Acidaminococcusfermentans was transformed by site-directed mutagenesis into an acyl-CoAhydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA(Mack and Buckel, FEBS Lett. 405:209-212 (1997)). This indicates thatthe enzymes encoding acetoacetyl-CoA transferases and4-hydroxybutyryl-CoA transferases can also be used as hydrolases withcertain mutations to change their function. The acetyl-CoA hydrolase,ACH1, from S. cerevisiae represents another candidate hydrolase (Buu etal., J. Biol. Chem. 278:17203-17209 (2003)). Information related tothese proteins and genes is shown below:

TABLE 20 Protein GENBANK ID GI NUMBER ORGANISM Acot12 NP_570103.118543355 Rattus norvegicus GctA CAA57199 559392 Acidaminococcusfermentans GctB CAA57200 559393 Acidaminococcus fermentans ACH1NP_009538 6319456 Saccharomyces cerevisiae

Another candidate hydrolase is the human dicarboxylic acid thioesterase,acot8, which exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA,sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J. Biol. Chem.280:38125-38132 (2005)) and the closest E. coli homolog, tesB, which canalso hydrolyze a broad range of CoA thioesters (Naggert et al., J. Biol.Chem. 266:11044-11050 (1991)) including 3-hydroxybutyryl-CoA (Tseng etal., Appl. Environ. Microbiol. 75(10):3137-3145 (2009)). A similarenzyme has also been characterized in the rat liver (Deana, Biochem.Int. 26:767-773 (1992)). Other potential E. coli thioester hydrolasesinclude the gene products of tesA (Bonner and Bloch, J. Biol. Chem.247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol. Rev.29:263-279 (2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)), paaI(Song et al., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB (Leduc etal., J. Bacteriol. 189:7112-7126 (2007)). Information related to theseproteins and genes is shown below:

TABLE 21 Protein GENBANK ID GI NUMBER ORGANISM Acot8 CAA15502 3191970Homo sapiens TesB NP_414986 16128437 Escherichia coli Acot8 NP_57011251036669 Rattus norvegicus TesA NP_415027 16128478 Escherichia coli YbgCNP_415264 16128711 Escherichia coli PaaI NP_415914 16129357 Escherichiacoli YbdB NP_415129 16128580 Escherichia coli

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J. Biol. Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., supra (1994); Shimomura et al., Methods Enzymol. 324:229-240(2000)) and Homo sapiens (Shimomura et al., supra (1994). Candidategenes by sequence homology include hibch of Saccharomyces cerevisiae andBC_(—)2292 of Bacillus cereus. BC_(—)2292 was shown to demonstrate3-hydroxybutyryl-CoA hydrolase activity and function as part of apathway for 3-hydroxybutyrate synthesis when engineered into Escherichiacoli (Lee et al., Appl. Microbiol. Biotechnol. 79:633-641 (2008)).Information related to these proteins and genes is shown below:

TABLE 22 GENBANK Protein ID GI NUMBER ORGANISM Hibch Q5XIE6.2 146324906Rattus norvegicus Hibch Q6NVY1.2 146324905 Homo sapiens Hibch P28817.22506374 Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacilluscereus ATCC 14579

The hydrolysis of acetoacetyl-CoA or 4-hydroxybutyryl-CoA canalternatively be carried out by a single enzyme or enzyme complex thatexhibits acetoacetyl-CoA or 4-hydroxybutyryl-CoA synthetase activity.This activity enables the net hydrolysis of the CoA-ester of eithermolecule, and in some cases, results in the simultaneous generation ofATP. For example, the product of the LSC1 and LSC2 genes of S.cerevisiae and the sucC and sucD genes of E. coli naturally form asuccinyl-CoA synthetase complex that catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Gruys et al., U.S. Pat. No.5,958,745, filed Sep. 28, 1999). Information related to these proteinsand genes is shown below:

TABLE 23 Protein GENBANK ID GI NUMBER ORGANISM SucC NP_415256.1 16128703Escherichia coli SucD AAC73823.1 1786949 Escherichia coli LSC1 NP_0147856324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomycescerevisiae

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical J. 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacilis subtilis (Boweretal., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidateenzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa etal., Biochim. Biophys. Acta 1779:414-419 (2008)) and Homo sapiens(Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003)), which naturallycatalyze the ATP-dependant conversion of acetoacetate intoacetoacetyl-CoA. 4-Hydroxybutyryl-CoA synthetase activity has beendemonstrated in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)). This function has been tentatively assigned tothe Msed_(—)1422 gene. Information related to these proteins and genesis shown below:

TABLE 24 GI Protein GENBANK ID NUMBER ORGANISM Phl CAJ15517.1 77019264Penicillium chrysogenum PhlB ABS19624.1 152002983 Penicilliumchrysogenum PaaF AAC24333.2 22711873 Pseudomonas putida BioW NP_390902.250812281 Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACSNP_076417.2 31982927 Homo sapiens Msed_1422 YP_001191504 146304188Metallosphaera sedula

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch. Microbiol.182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra(2004)). The enzymes from A. fulgidus, H. marismortui and P. aerophilumhave all been cloned, functionally expressed, and characterized in E.coli (Musfeldt et al., supra; Brasen et al., supra (2004)). Informationrelated to these proteins and genes is shown below:

TABLE 25 Protein GENBANK ID GI NUMBER ORGANISM AF1211 NP_070039.111498810 Archaeoglobus fulgidus DSM 4304 Scs YP_135572.1 55377722Haloarcula marismortui ATCC 43049 PAE3250 NP_560604.1 18313937Pyrobaculum aerophilum str. IM2

An alternative method for removing the CoA moiety from acetoacetyl-CoAor 4-hydroxybutyryl-CoA is to apply a pair of enzymes such as aphosphate-transferring acyltransferase and a kinase to impartacetoacetyl-CoA or 4-hydroxybutyryl-CoA synthetase activity. Exemplarynames for these enzymes includephosphotrans-4-hydroxybutyrylase/4-hydroxybutyrate kinase, which canremove the CoA moiety from 4-hydroxybutyryl-CoA, andphosphotransacetoacetylase/acetoacetate kinase which can remove the CoAmoiety from acetoacetyl-CoA. This general activity enables the nethydrolysis of the CoA-ester of either molecule with the simultaneousgeneration of ATP. For example, the butyrate kinase(buk)/phosphotransbutyrylase (ptb) system from Clostridiumacetobutylicum has been successfully applied to remove the CoA groupfrom 3-hydroxybutyryl-CoA when functioning as part of a pathway for3-hydroxybutyrate synthesis (Tseng et al., Appl. Environ. Microbiol.75(10):3137-3145 (2009)). Specifically, the ptb gene from C.acetobutylicum encodes an enzyme that can convert an acyl-CoA into anacyl-phosphate (Walter et al. Gene 134(1): p. 107-11 (1993)); Huang etal. J Mol Microbiol Biotechnol 2(1): p. 33-38 (2000). Additional ptbgenes can be found in butyrate-producing bacterium L2-50 (Louis et al.J. Bacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez etal. Curr. Microbiol 42:345-349 (2001)). Additional exemplaryphosphate-transferring acyltransferases include phosphotransacetylase,encoded by pta. The pta gene from E. coli encodes an enzyme that canconvert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilizepropionyl-CoA instead of acetyl-CoA forming propionate in the process(Hes slinger et al. Mol. Microbiol 27:477-492 (1998)). Informationrelated to these proteins and genes is shown below:

TABLE 26 Protein GENBANK ID GI NUMBER ORGANISM Pta NP_416800.1 16130232Escherichia coli Ptb NP_349676 15896327 Clostridium acetobutylicum PtbAAR19757.1 38425288 butyrate-producing bacterium L2-50 Ptb CAC07932.110046659 Bacillus megaterium

Exemplary kinases include the E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)), the C.acetobutylicum butyrate kinases, encoded by buk1 and buk2 ((Walter etal. Gene 134(1):107-111 (1993); Huang et al. J. Mol. Microbiol.Biotechnol. 2(1):33-38 (2000)), and the E. coli gamma-glutamyl kinase,encoded by proB (Smith et al. J. Bacteriol. 157:545-551 (1984)). Theseenzymes phosphorylate acetate, butyrate, and glutamate, respectively.The ackA gene product from E. coli also phosphorylates propionate(Hesslinger et al. Mol. Microbiol. 27:477-492 (1998)). Informationrelated to these proteins and genes is shown below:

TABLE 27 Protein GENBANK ID GI NUMBER ORGANISM AckA NP_416799.1 16130231Escherichia coli Buk1 NP_349675 15896326 Clostridium acetobutylicum Buk2Q97II1 20137415 Clostridium acetobutylicum ProB NP_414777.1 16128228Escherichia coli

Acetoacetate decarboxylase converts acetoacetate into carbon dioxide andacetone. Exemplary acetoacetate decarboxylase enzymes are encoded by thegene products of adc from C. acetobutylicum (Petersen and Bennett, ApplEnviron. Microbiol 56:3491-3498 (1990)) and adc from Clostridiumsaccharoperbutylacetonicum (Kosaka, et al., Biosci. Biotechnol Biochem.71:58-68 (2007)). The enzyme from C. beijerinkii can be inferred fromsequence similarity. These proteins are identified below in Table 28.

TABLE 28 Protein GenBank ID GI Number Organism Adc NP_149328.1 15004868Clostridium acetobutylicum Adc AAP42566.1 31075386 Clostridiumsaccharoperbutylacetonicum Adc YP_001310906.1 150018652 Clostridiumbeijerinckii

The final step in the isopropanol synthesis pathway involves thereduction of acetone to isopropanol. Exemplary alcohol dehydrogenaseenzymes capable of this transformation include adh from C. beijerinckii(Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007); Jojima etal., Appl. Microbiol. Biotechnol. 77:1219-1224 (2008)) and adh fromThermoanaerobacter brockii (Hanai et al., Appl. Environ. Microbiol.73:7814-7818 (2007); Peretz et al., Anaerobe 3:259-270 (1997)).Additional characterized enzymes include alcohol dehydrogenases fromRalstonia eutropha (formerly Alcaligenes eutrophus) (Steinbuchel andSchlegel et al., Eur. J. Biochem. 141:555-564 (1984)) and Phytomonasspecies (Uttaro and Opperdoes et al., Mol. Biochem. Parasitol.85:213-219 (1997)).

TABLE 29 Protein GenBank ID GI Number Organism Adh P14941.1 113443Thermoanaerobobacter brockii Adh AAA23199.2 60592974 Clostridiumbeijerinckii Adh YP_299391.1 73539024 Ralstonia eutropha iPDH AAP39869.131322946 Phtomonas sp.

Exemplary 3-hydroxyacyl dehydrogenases which convert acetoacetyl-CoA to3-hydroxybutyryl-CoA include hbd from C. acetobutylicum (Boynton et al.,J. Bacteriol. 178:3015-3024 (1996)), hbd from C. beijerinckii (Colby andChen et al., Appl. Environ. Microbiol. 58:3297-3302 (1992)), and anumber of similar enzymes from Metallosphaera sedula (Berg et al.,Science 318:1782-1786 (2007)).

TABLE 29 Protein GenBank ID GI Number Organism hbd NP_349314.1 15895965Clostridium acetobutylicum hbd AAM14586.1 20162442 Clostridiumbeijerinckii Msed_1423 YP_001191505 146304189 Metallosphaera sedulaMsed_0399 YP_001190500 146303184 Metallosphaera sedula Msed_0389YP_001190490 146303174 Metallosphaera sedula Msed_1993 YP_001192057146304741 Metallosphaera sedula

The gene product of crt from C. acetobutylicum catalyzes the dehydrationof 3-hydroxybutyryl-CoA to crotonyl-CoA (Atsumi et al., Metab. Eng.(2007); Boynton et al., J. Bacteriol. 178:3015-3024 (1996)). Further,enoyl-CoA hydratases are reversible enzymes and thus suitable candidatesfor catalyzing the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA.The enoyl-CoA hydratases, phaA and phaB, of P. putida are believed tocarry out the hydroxylation of double bonds during phenylacetatecatabolism (Olivera et al., Proc. Nat. Acad. Sci. U.S.A. 95:6419-6424(1998)). The paaA and paaB from P. fluorescens catalyze analogoustransformations (Olivera et al., Proc. Nat. Acad. Sci. U.S.A.95:6419-6424 (1998)). Lastly, a number of Escherichia coli genes havebeen shown to demonstrate enoyl-CoA hydratase functionality includingmaoC, paaF, and paaG (Ismail et al., Eur. J. Biochem. 270:3047-3054(2003); Park and Lee, J. Bacteriol. 185:5391-5397 (2003); Park and Lee,Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park and Yup,Biotechnol. Bioeng. 86:681-686 (2004)).

TABLE 30 Protein GenBank ID  GI Number Organism crt NP_349318.1 15895969Clostridium acetobutylicum paaA NP_745427.1 26990002 Pseudomonas putidapaaB NP_745426.1 26990001 Pseudomonas putida phaA ABF82233.1 106636093Pseudomonas fluorescens phaB ABF82234.1 106636094 Pseudomonasfluorescens maoC NP_415905.1 16129348 Escherichia coli paaF NP_415911.116129354 Escherichia coli paaG NP_415912.1 16129355 Escherichia coli

Several enzymes that naturally catalyze the reverse reaction (i.e., thedehydration of 4-hydroxybutyryl-CoA to crotonoyl-CoA) in vivo have beenidentified in numerous species. This transformation is used for4-aminobutyrate fermentation by Clostridium aminobutyricum (Scherf andBuckel, Eur. J. Biochem. 215:421-429 (1993)) and succinate-ethanolfermentation by Clostridium kluyveri (Scherf et al., Arch. Microbiol.161:239-245 (1994)). The transformation is also a step in Archaea, forexample, Metallosphaera sedula, as part of the3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxideassimilation pathway (Berg et al., Science 318:1782-1786 (2007)). Thispathway uses the hydration of crotonoyl-CoA to form4-hydroxybutyryl-CoA. The reversibility of 4-hydroxybutyryl-CoAdehydratase is well-documented (Friedrich et al., Angew. Chem. Int. Ed.Engl. 47:3254-3257 (2008); Muh et al., Eur. J. Biochem. 248:380-384(1997); Muh et al., Biochemistry 35:11710-11718 (1996)) and theequilibrium constant has been reported to be about 4 on the side ofcrotonoyl-CoA (Scherf and Buckel, Eur. J. Biochem. 215:421-429 (1993)).This indicates that the downstream 4-hydroxybutyryl-CoA dehydrogenasekeeps the 4-hydroxybutyryl-CoA concentration low so as to not create athermodynamic bottleneck at crotonyl-CoA.

TABLE 31 Protein GenBank ID GI Number Organism AbfD CAB60035 70910046Clostridium aminobutyricum AbfD YP_001396399 153955634 Clostridiumkluyveri Msed_1321 YP_001191403 146304087 Metallosphaera sedulaMsed_1220 YP_001191305 146303989 Metallosphaera sedula

Alcohol-forming 4-hydroxybutyryl-CoA reductase enzymes catalyze the 2reduction steps required to form 1,4-butanediol from4-hydroxybutyryl-CoA. Exemplary 2-step oxidoreductases that convert anacyl-CoA to alcohol include those that transform substrates such asacetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBS.Lett. 281:59-63 (1991)) and butyryl-CoA to butanol (e.g. adhE2 from C.acetobutylicum (Fontaine et al., J. Bacteriol. 184:821-830 (2002)). TheadhE2 enzyme from C. acetobutylicum was specifically shown in ref. Burket al., WO/2008/115840 (2008). to produce BDO from 4-hydroxybutyryl-CoA.In addition to reducing acetyl-CoA to ethanol, the enzyme encoded byadhE in Leuconostoc mesenteroides has been shown to oxide the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J.Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol. Lett.27:505-510 (2005)).

TABLE 32 Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 55818563 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. AnNADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410(2000); Strauss and Fuchs, Eur. J. Biochem. 215:633-643 (1993)). Thisenzyme, with a mass of 300 kDa, is highly substrate-specific and showslittle sequence similarity to other known oxidoreductases (Hugler etal., J. Bacteriol. 184:2404-2410 (2002)). No enzymes in other organismshave been shown to catalyze this specific reaction; however there isbioinformatic evidence that other organisms may have similar pathways(Klatt et al., Environ. Microbiol. 9:2067-2078 (2007)). Enzymecandidates in other organisms including Roseiflexus castenholzii,Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can beinferred by sequence similarity.

TABLE 33 Protein GenBank ID GI Number Organism mcr AAS20429.1 42561982Chloroflexus aurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexuscastenholzii NAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1MGP2080_00535 ZP_01626393.1 119504313 marine gamma proteobacteriumHTCC2080

An alternative route to BDO from 4-hydroxybutyryl-CoA involves firstreducing this compound to 4-hydroxybutanal. Several acyl-CoAdehydrogenases are capable of reducing an acyl-CoA to its correspondingaldehyde. Exemplary genes that encode such enzymes include theAcinetobacter calcoaceticus acrl encoding a fatty acyl-CoA reductase(Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997)), theAcinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependentsuccinate semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)). SucD of P. gingivalis is another succinate semialdehydedehydrogenase (Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)).These succinate semialdehyde dehydrogenases were specifically shown inref. Burk et al., WO/2008/115840 (2008) to convert 4-hydroxybutyryl-CoAto 4-hydroxybutanal as part of a pathway to produce 1,4-butanediol. Theenzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encodedby bphG, is yet another capable enzyme as it has been demonstrated tooxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde,isobutyraldehyde and formaldehyde (Powlowski et al., J. Bacteriol.175:377-385 (1993)).

TABLE 34 Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 sucD P38947.1172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archael bacteria (Berg et al., Science 318:1782-1786(2007); Thauer, R. K. Science 318:1732-1733 (2007)). The enzyme utilizesNADPH as a cofactor and has been characterized in Metallosphaera andSulfolobus spp (Alber et al., J. Bacteriol. 188:8551-8559 (2006); Hugleret al., J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded byMsed_(—)0709 in Metallosphaera sedula (Alber et al., J. Bacteriol.188:8551-8559 (2006); Berg et al., Science 318:1782-1786 (2007)). A geneencoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al., J. Bacteriol.188:8551-8559 (2006)). Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius. Yet another gene forCoA-acylating aldehyde dehydrogenase is the ald gene from Clostridiumbeijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). Thisenzyme has been reported to reduce acetyl-CoA and butyryl-CoA to theircorresponding aldehydes. This gene is very similar to eutE that encodesacetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth,Appl. Environ. Microbiol. 65:4973-4980 (1999). These proteins areidentified below in Table 35.

TABLE 35 GI Protein GenBank ID Number Organism Msed_0709 YP_001190808.1146303492 Metallosphaera sedula Mcr NP_378167.1 15922498 Sulfolobustokodaii asd-2 NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370YP_256941.1 70608071 Sulfolobus acidocaldarius Ald AAT66436 49473535Clostridium beijerinckii eutE AAA80209 687645 Salmonella typhimuriumeutE P77445 2498347 Escherichia coli

4-Hydroxybutyryl-CoA can also be converted to 4-hydroxybutanal inseveral enzymatic steps, though the intermediate 4-hydroxybutyrate.First, 4-hydroxybutyryl-CoA can be converted to 4-hydroxybutyrate by aCoA transferase, hydrolase or synthetase. Alternately,4-hydroxybutyryl-CoA can be converted to 4-hydroxybutyrate via aphosphonated intermediate by enzymes withphosphotrans-4-hydroxybutyrylase and 4-hydroxybutyrate kinase. Exemplarycandidates for these enzymes are described above.

Subsequent conversion of 4-hydroxybutyrate to 4-hydroxybutanal iscatalyzed by an aryl-aldehyde dehydrogenase, or equivalently acarboxylic acid reductase. Such an enzyme is found in Nocardia iowensis.Carboxylic acid reductase catalyzes the magnesium, ATP andNADPH-dependent reduction of carboxylic acids to their correspondingaldehydes (Venkitasubramanian et al., J. Biol. Chem. 282:478-485 (2007))and is capable of catalyzing the conversion of 4-hydroxybutyrate to4-hydroxybutanal. This enzyme, encoded by car, was cloned andfunctionally expressed in E. coli (Venkitasubramanian et al., J. Biol.Chem. 282:478-485 (2007)). Expression of the npt gene product improvedactivity of the enzyme via post-transcriptional modification. The nptgene encodes a specific phosphopantetheine transferase (PPTase) thatconverts the inactive apo-enzyme to the active holo-enzyme. The naturalsubstrate of this enzyme is vanillic acid, and the enzyme exhibits broadacceptance of aromatic and aliphatic substrates (Venkitasubramanian etal., in Biocatalysis in the Pharmaceutical and Biotechnology Industires,ed. R. N. Patel, Chapter 15, pp. 425-440, CRC Press LLC, Boca Raton,Fla. (2006)).

GenBank Gene Accession name GI No. No. Organism Car 40796035 AAR91681.1Nocardia iowensis (sp. NRRL 5646) Npt 114848891 ABI83656.1 Nocardiaiowensis (sp. NRRL 5646)

Additional car and npt genes can be identified based on sequencehomology.

GenBank Gene name GI No. Accession No. Organism fadD9 121638475YP_978699.1 Mycobacterium bovis BCG BCG_2812c 121638674 YP_978898.1Mycobacterium bovis BCG nfa20150 54023983 YP_118225.1 Nocardia farcinicaIFM 10152 nfa40540 54026024 YP_120266.1 Nocardia farcinica IFM 10152SGR_6790 182440583 YP_001828302.1 Streptomyces griseus subsp. griseusNBRC 13350 SGR_665 182434458 YP_001822177.1 Streptomyces griseus subsp.griseus NBRC 13350 MSMEG_2956 YP_887275.1 YP_887275.1 Mycobacteriumsmegmatis MC2 155 MSMEG_5739 YP_889972.1 118469671 Mycobacteriumsmegmatis MC2 155 MSMEG_2648 YP_886985.1 118471293 Mycobacteriumsmegmatis MC2 155 MAP1040c NP_959974.1 41407138 Mycobacterium aviumsubsp. paratuberculosis K-10 MAP2899c NP_961833.1 41408997 Mycobacteriumavium subsp. paratuberculosis K-10 MMAR_2117 YP_001850422.1 183982131Mycobacterium marinum M MMAR_2936 YP_001851230.1 183982939 Mycobacteriummarinum M MMAR_1916 YP_001850220.1 183981929 Mycobacterium marinum MTpauDRAFT_33060 ZP_04027864.1 227980601 Tsukamurella paurometabola DSM20162 TpauDRAFT_20920 ZP_04026660.1 ZP_04026660.1 Tsukamurellapaurometabola DSM 20162 CPCC7001_1320 ZP_05045132.1 254431429 CyanobiumPCC7001 DDBDRAFT_0187729 XP_636931.1 66806417 Dictyostelium discoideumAX4

An additional enzyme candidate found in Streptomyces griseus is encodedby the griC and griD genes. This enzyme is believed to convert3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde asdeletion of either griC or griD led to accumulation of extracellular3-acetylamino-4-hydroxybenzoic acid, a shunt product of3-amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J. Antibiot.60(6):380-387 (2007)). Co-expression of griC and griD with SGR_(—)665,an enzyme similar in sequence to the Nocardia iowensis npt, can bebeneficial.

Gene GenBank name GI No. Accession No. Organism griC 182438036YP_001825755.1 Streptomyces griseus subsp. griseus NBRC 13350 Grid182438037 YP_001825756.1 Streptomyces griseus subsp. griseus NBRC 13350

An enzyme with similar characteristics, alpha-aminoadipate reductase(AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in somefungal species. This enzyme naturally reduces alpha-aminoadipate toalpha-aminoadipate semialdehyde. The carboxyl group is first activatedthrough the ATP-dependent formation of an adenylate that is then reducedby NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizesmagnesium and requires activation by a PPTase. Enzyme candidates for AARand its corresponding PPTase are found in Saccharomyces cerevisiae(Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al.,Mol. Genet. Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe(Ford et al., Curr. Genet. 28:131-137 (1995)). The AAR from S. pombeexhibited significant activity when expressed in E. coli (Guo et al.,Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum acceptsS-carboxymethyl-L-cysteine as an alternate substrate, but did not reactwith adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J.Biol. Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenumPPTase has not been identified to date.

Gene GenBank name GI No. Accession No. Organism LYS2 171867 AAA34747.1Saccharomyces cerevisiae LYS5 1708896 P50113.1 Saccharomyces cerevisiaeLYS2 2853226 AAC02241.1 Candida albicans LYS5 28136195 AAO26020.1Candida albicans Lys1p 13124791 P40976.3 Schizosaccharomyces pombe Lys7p1723561 Q10474.1 Schizosaccharomyces pombe Lys2 3282044 CAA74300.1Penicillium chrysogenum

Enzymes exhibiting 1,4-butanediol dehydrogenase activity are capable offorming 1,4-butanediol from 4-hydroxybutanal. Exemplary genes encodingenzymes that catalyze the conversion of an aldehyde to alcohol (i.e.,alcohol dehydrogenase or equivalently aldehyde reductase) include alrAencoding a medium-chain alcohol dehydrogenase for C₂-C₁₄ (Tani et al.,Appl. Environ. Microbiol. 66:5231-5235 (2000)), ADH2 from Saccharomycescerevisiae Atsumi et al. Nature 451:86-89 (2008)), yqhD from E. coliwhich has preference for molecules longer than C(3) (Sulzenbacher etal., J. Mol. Biol. 342:489-502 (2004)), and bdh I and bdh II from C.acetobutylicum which converts butyryaldehyde into butanol (Walter etal., J. Bacteriol. 174:7149-7158 (1992)). ADH1 from Zymomonas mobilishas been demonstrated to have activity on a number of aldehydesincluding formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde,and acrolein (Kinoshita, Appl. Microbiol. Biotechnol. 22:249-254(1985)).

TABLE 36 Protein GenBank ID GI Number Organism alrA BAB12273.1 9967138Acinetobacter sp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharymycescerevisiae yqhD NP_417484.1 16130909 Escherichia coli bdh I NP_349892.115896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al., J. Forensic Sci.49:379-387 (2004)), Clostridium kluyveri (Wolff and Kenealy, ProteinExpr. Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz etal., J. Biol. Chem. 278:41552-41556 (2003)).

TABLE 37 Protein GenBank ID GI Number Organism 4hbd YP_726053.1113867564 Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridiumkluyveri DSM 555 4hbd Q94B07 75249805 Arabidopsis thaliana

The first step in the cloning and expression process is to express in E.coli the minimal set of genes (e.g., MtaA, MtaB, and MtaC) necessary toproduce Me-THF from methanol. These methyltransferase activities useCoenzyme B₁₂ (cobalamin) as a cofactor. In Moorella thermoacetica, acascade of methyltransferase proteins mediate incorporation of methanolderived methyl groups into the acetyl-CoA synthase pathway. Recent work(Das et al., Proteins 67:167-176 (2007)) indicates that MtaABC areencoded by Moth_(—)1208-09 and Moth_(—)2346. These genes are cloned viaproof-reading PCR and linked together for expression in a high-copynumber vector such as pZE22-S under control of the repressible PA1-lacO1promoter (Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997)).Cloned genes are verified by PCR and or restriction enzyme mapping todemonstrate construction and insertion of the 3-gene set into theexpression vector. DNA sequencing of the presumptive clones is carriedout to confirm the expected sequences of each gene. Once confirmed, thefinal construct is expressed in E. coli K-12 (MG1655) cells by additionof IPTG inducer between 0.05 and 1 mM final concentration. Expression ofthe cloned genes is monitored using SDS-PAGE of whole cell extracts. Tooptimize levels of soluble vs. pellet (potentially inclusion bodyorigin) protein, the affect of titration of the promoter on these levelscan be examined. If no acceptable expression is obtained, higher orlower copy number vectors or variants in promoter strength are tested.

To determine if expression of the MtaABC proteins from M. thermoaceticaconfers upon E. coli the ability to transfer methyl groups from methanolto tetrahydrofolate (THF) the recombinant strain is fed methanol atvarious concentrations. Activity of the methyltransferase system isassayed anaerobically as described for vanillate as a methyl source inM. thermoacetica (Naidu and Ragsdale, J. Bacteriol. 183:3276-3281(2001)) or for Methanosarcina barkeri methanol methyltransferase (Saueret al., Eur. J. Biochem. 243:670-677 (1997); Tallant and Krzycki, J.Bacteriol. 178:1295-1301 (1996); Tallant and Krzycki, J. Bacteriol.179:6902-6911 (1997); Tallant et al., J. Biol. Chem. 276:4485-4493(2001)). For a positive control, M. thermoacetica cells are cultured inparallel and assayed anaerobically to confirm endogenousmethyltransferase activity. Demonstration of dependence on exogenouslyadded coenzyme B₁₂ confirms methanol:corrinoid methyltransferaseactivity in E. coli.

Once methyltransferase expression is achieved, further work is performedtowards optimizing the expression. Titrating the promoter in theexpression vector enables the testing of a range of expression levels.This is then used as a guide towards the expression required insingle-copy, or enables the determination of whether or not asingle-copy of these genes allows sufficient expression. If so, themethyltransferase genes are integrated into the chromosome as a single,synthetic operon. This entails targeted integration using RecET-based‘recombineering’ (Angrand et al., Nucleic Acids Res. 27:e16 (1999);Muyrers et al., Nucleic Acids Res. 27:1555-1557 (1999); Zhang et al.,1998 Nat. Genet. 20:123-128 (1998)). A potential issue with RecET-basedintegration of a cassette and removal of a FRT or loxP-boundedselectable marker by FLP or Cre is the production of a recombinationscar at each integration site. While problems caused by this can beminimized by a number of methods, other means that do not leave genomicscars are available. The standard alternative, is to introduce thedesired genes using integrative ‘suicide’ plasmids coupled tocounter-selection such as that allowed by the Bacillus sacB gene (Linket al., J. Bacteriol. 179:6228-6237 (1997)); in this way, markerless andscar less insertions at any location in the E. coli chromosome can begenerated. The final goal is a strain of E. coli K-12 expressingmethanol:corrinoid methyltransferase activity under an induciblepromoter and in single copy (chromosomally integrated).

Using standard PCR methods, entire ACS/CODH operons are assembled intolow or medium copy number vectors such as pZA33-S (P15A-based) orpZS13-S (pSC101-based). As described for the methyltransferase genes,the structure and sequence of the cloned genes are confirmed. Expressionis monitored via protein gel electrophoresis of whole-cell lysates grownunder strictly anaerobic conditions with the requisite metals (Ni, Zn,Fe) and coenzyme B₁₂ provided. As necessary, the gene cluster ismodified for E. coli expression by identification and removal of anyapparent terminators and introduction of consensus ribosomal bindingsites chosen from sites known to be effective in E. coli (Barrick etal., Nucleic Acids Res. 22:1287-1295 (1994); Ringquist et al., Mol.Microbiol. 6:1219-1229 (1992)). However, each gene cluster is cloned andexpressed in a manner parallel to its native structure and expression.This helps ensure the desired stoichiometry between the various geneproducts—most of which interact with each other. Once satisfactoryexpression of the CODH/ACS gene cluster under anaerobic conditions isachieved, the ability of cells expressing these genes to fix CO and/orCO₂ into cellular carbon is assayed. Initial conditions employ strictlyanaerobically grown cells provided with exogenous glucose as a carbonand energy source via substrate-level phosphorylation or anaerobicrespiration with nitrate as an electron acceptor. Additionally,exogenously provided CH₃-THF is added to the medium.

The ACS/CODH genes are cloned and expressed in cells also expressing themethanol-methyltransferase system. This is achieved by introduction ofcompatible plasmids expressing ACS/CODH into MTR-expressing cells. Foradded long-term stability, the ACS/CODH and MTR genes can also beintegrated into the chromosome. After strains of E. coli capable ofutilizing methanol to produce Me-THF and of expressing active CODH/ACSgene are made, they are assayed for the ability to utilize both methanoland syngas for incorporation into acetyl-CoA, acetate, and cell mass.Initial conditions employ strictly anaerobically grown cells providedwith exogenous glucose as a carbon and energy source. Alternatively, orin addition to glucose, nitrate will be added to the fermentation brothto serve as an electron acceptor and initiator of growth. Anaerobicgrowth of E. coli on fatty acids, which are ultimately metabolized toacetyl-CoA, has been demonstrated in the presence of nitrate (Campbellet al., Mol. Microbiol. 47:793-805 (2003)). Oxygen can also be providedas long as its intracellular levels are maintained below any inhibitionthreshold of the engineered enzymes. ‘Synthetic syngas’ of a compositionsuitable for these experiments is employed along with methanol.¹³C-labeled methanol or ¹³C-labeled CO are provided to the cells andanalytical mass spectrometry is employed to measure incorporation of thelabeled carbon into acetate and cell mass (e.g., proteinogenic aminoacids).

The pyruvate ferredoxin oxidoreductase genes from M. thermoacetica, D.africanus, and E. coli are cloned and expressed in strains exhibitingMTR and ACS/CODH activities. Conditions, promoters, etc., are describedabove. Given the large size of the PFOR genes and oxygen sensitivity ofthe corresponding enzymes, tests will be performed using low orsingle-copy plasmid vectors or single-copy chromosomal integrations.Activity assays described in ref. (Furdui and Ragsdale, J. Biol. Chem.275:28494-28499 (2000)) will be applied to demonstrate activity. Inaddition, demonstration of growth on the gaseous carbon sources andmethanol in the absence of an external electron acceptor will providefurther evidence for PFOR activity in vivo.

The endogenous hydrogen-utilizing hydrogenase activity of the hostorganism is tested by growing the cells as described above in thepresence and absence of hydrogen. If a dramatic shift towards theformation of more reduced products during fermentation is observed(e.g., increased ethanol as opposed to acetate), this indicates thatendogenous hydrogenase activity is sufficiently active. In this case, noheterologous hydrogenases are cloned and expressed. If the nativeenzymes do not have sufficient activity or reduce the needed acceptor,the genes encoding an individual hydrogenase complex are cloned andexpressed in strains exhibiting MTR, ACS/CODH, and PFOR activities.Conditions, promoters, etc., are described above.

The normative genes needed for isopropanol synthesis are cloned onexpression plasmids as described previously. The host strain alsoexpresses methanol methyltransferase activity, CODH/ACS activity, andpossibly PFOR and hydrogenase activities. At this point, these(CODH/ACS, etc.) genes are integrated into the genome and expressed frompromoters that can be used constitutively or with inducers (i.e.,PA1-lacO1 is inducible in cells containing lad or is otherwiseconstitutive). Once expression and yields of isopropanol are optimized,the base strain is further modified by integration of a single copy ofthese genes at a neutral locus. Given the relatively limited number ofgenes (at minimum, 3, and at most, 6), Applicants construct anartificial operon encoding the required genes. This operon is introducedusing integrative plasmids and is coupled to counter-selection methodssuch as that allowed by the Bacillus sacB gene (Link et al., J.Bacteriol. 179:6228-6237 (1997)). In this way, markerless and scar lessinsertions at any location in the E. coli chromosome can be generated.Optimization involves altering gene order as well as ribosomal bindingsites and promoters.

To over express any native genes, for example, the native atoB (b2224)gene of E. coli which can serve as an alternative to the C.acetobutylicum acetyl-coenzyme A [CoA] acetyltransferase required forisopropanol production, RecET-based methods are applied to integrate astronger upstream promoter. In the case of atoB, this gene is the lastin an operon and the next gene downstream (yfaP) is both non-essentialand in the opposite orientation. Therefore, polarity should not be anissue. A cassette containing a selectable marker such as spectinomycinresistance or chloramphenicol resistance flanked by FRT or loxP sites isused to select for introduction of a strong constitutive promoter (e.g.,pL). Once the correct clone is obtained and validated, using qRT-PCR,FLP or Cre expression is used to select for removal of the FRT- orloxP-bounded marker.

The normative genes needed for 4-hydroxybutyrate synthesis are cloned onexpression plasmids as described previously. The host strain alsoexpresses methanol methyltransferase activity, CODH/ACS activity, andpossibly PFOR and hydrogenase activities. At this point, these(CODH/ACS, etc.) genes are integrated into the genome and expressed frompromoters that can be used constitutively or with inducers (i.e.,PA1-lacO1 is inducible in cells containing lad or is otherwiseconstitutive). Once expression and yields of 4-hydroxybutyrate areoptimized, the base strain is further modified by integration of asingle copy of these genes at a neutral locus. Given the relativelylimited number of genes (at minimum, 5, and at most, 6), an artificialoperon encoding the required genes can be constructed. This operon isintroduced using integrative plasmids and is coupled tocounter-selection methods such as that allowed by the Bacillus sacB gene(Link et al., J. Bacteriol. 179:6228-6237 (1997)). In this way,markerless and scar less insertions at any location in the E. colichromosome can be generated. Optimization involves altering gene orderas well as ribosomal binding sites and promoters.

The normative genes needed for 1,4-butanediol synthesis are cloned onexpression plasmids as described previously. The host strain alsoexpresses methanol methyltransferase activity, CODH/ACS activity, andpossibly PFOR and hydrogenase activities. At this point, these(CODH/ACS, etc.) genes are integrated into the genome and expressed frompromoters that can be used constitutively or with inducers (i.e.,PA1-lacO1 is inducible in cells containing lad or is otherwiseconstitutive). Once expression and yields of 1,4-butanediol areoptimized, the base strain is further modified by integration of asingle copy of these genes at a neutral locus. Given the relativelylimited number of genes (at minimum, 5, and at most, 6), an artificialoperon encoding the required genes can be constructed. This operon isintroduced using integrative plasmids and is coupled tocounter-selection methods such as that allowed by the Bacillus sacB gene(Link et al., J. Bacteriol. 179:6228-6237 (1997)). In this way,markerless and scar less insertions at any location in the E. colichromosome can be generated. Optimization involves altering gene orderas well as ribosomal binding sites and promoters.

Engineering the capability to convert synthesis gas into acetyl-CoA, thecentral metabolite from which all cell mass components and many valuableproducts can be derived, into a foreign host such as E. coli can beaccomplished following the expression of exogenous genes that encodevarious proteins of the Wood-Ljungdahl pathway. This pathway is highlyactive in acetogenic organisms such as Moorella thermoacetica (formerly,Clostridium thermoaceticum), which has been the model organism forelucidating the Wood-Ljungdahl pathway since its isolation in 1942(Fontaine et al., J. Bacteriol. 43:701-715 (1942)). The Wood-Ljungdahlpathway comprises of two branches: the Eastern (or methyl) branch thatenables the conversion of CO₂ to methyltetrahydrofolate (Me-THF) and theWestern (or carbonyl) branch that enables the conversion of methyl-THF,CO, and Coenzyme-A into acetyl-CoA (FIG. 3). In some embodiments, thepresent invention provides a non-naturally occurring microorganismexpressing genes encoding enzymes that catalyze the methyl and carbonylbranches of the Wood-Ljungdahl pathway. Such an organism is capable ofconverting CO, CO₂, and/or H₂ into acetyl-CoA, cell mass, and products.

Another organism of the present invention contains three capabilitieswhich are depicted in FIG. 3A: 1) a functional methyl branch of theWood-Ljungdahl pathway which enables the conversion of THF and CO₂ to5-methyl-tetrahydrofolate, 2) the ability to combine CO, Coenzyme A, andthe methyl group of Me-THF to form acetyl-CoA, and 3) the ability tosynthesize isopropanol from acetyl-CoA. The fifth organism described inthis invention, depicted in FIG. 3B, contains a functional methyl branchof the Wood-Ljungdahl pathway, the ability to synthesize acetyl-CoA, andthe ability to synthesize 4-hydroxybutyrate from acetyl-CoA. The sixthorganism described in this invention, depicted in FIG. 3C, contains afunctional methyl branch of the Wood-Ljungdahl pathway, the ability tosynthesize acetyl-CoA, and the ability to synthesize 1,4-butanediol fromacetyl-CoA.

These three organisms are able to ‘fix’ carbon from exogenous CO and/orCO₂ to synthesize acetyl-CoA, cell mass, and products. A host organismengineered with these capabilities that also naturally possesses thecapability for anapleurosis (e.g., E. coli) can grow on thesyngas-generated acetyl-CoA in the presence of a suitable externalelectron acceptor such as nitrate. This electron acceptor is required toaccept electrons from the reduced quinone formed via succinatedehydrogenase. A further advantage of adding an external electronacceptor is that additional energy for cell growth, maintenance, andproduct formation can be generated from respiration of acetyl-CoA. Analternative strategy involves engineering a pyruvate ferredoxinoxidoreductase (PFOR) enzyme into the strain to enable synthesis ofbiomass precursors in the absence of an external electron acceptor. Afurther characteristic of the engineered organism is the capability forextracting reducing equivalents from molecular hydrogen. This enables ahigh yield of reduced products such as ethanol, butanol, isobutanol,isopropanol, 1,4-butanediol, succinic acid, fumaric acid, malic acid,4-hydroxybutyric acid, 3-hydroxypropionic acid, lactic acid, adipicacid, 6-aminocaproic acid, hexamethylenediamine, 2-hydroxyisobutyricacid, 3-hydroxyisobutyric acid, methacrylic acid, and acrylic acid.

The organisms provided herein can produce acetyl-CoA, cell mass, andtargeted chemicals, more specifically isopropanol, 4-hydroxybutyrate,and 1,4-butanediol, from: 1) CO, 2) CO₂ and H₂, 3) CO, CO₂, and H₂, 4)synthesis gas comprising CO and H₂, 5) synthesis gas comprising CO, CO₂,and H₂, 6) one or more carbohydrates, 7) methanol and one or morecarbohyrates, and 8) methanol. Exemplary carbohydrates include but arenot limited to glucose, sucrose, xylose, arabinose and glycerol.

Successfully engineering any of these pathways into an organism involvesidentifying an appropriate set of enzymes, cloning their correspondinggenes into a production host, optimizing the stability and expression ofthese genes, optimizing fermentation conditions, and assaying forproduct formation following fermentation. Below are described enzymesthat catalyze steps 1 through 5 of the pathways depicted in FIGS. 3Athrough 3C. These enzymes are required to enable the conversion ofsynthesis gas to acetyl-CoA. Enzymes for steps 6 through 17 in FIG. 3Aand steps 6 through 20 in FIGS. 3B and 3C were described above. Toengineer a production host for the utilization of syngas, one or moreexogenous DNA sequence(s) encoding the requisite enzymes can beexpressed in the microorganism.

Formate dehydrogenase is a two subunit selenocysteine-containing proteinthat catalyzes the incorporation of CO₂ into formate in Moorellathermoacetica (Andreesen and Ljungdahl, J. Bacteriol. 116:867-873(1973); Li et al., J. Bacteriol. 92:405-412 (1966); Yamamoto et al., J.Biol. Chem. 258:1826-1832 (1983). The loci, Moth_(—)2312 is responsiblefor encoding the alpha subunit of formate dehydrogenase while the betasubunit is encoded by Moth_(—)2314 (Pierce et al., Environ. Microbiol.(2008)). Another set of genes encoding formate dehydrogenase activitywith a propensity for CO₂ reduction is encoded by Sfum_(—)2703 throughSfum_(—)2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur. J.Biochem. 270:2476-2485 (2003)); Reda et al., Proc. Natl. Acad. Sci.U.S.A. 105:10654-10658 (2008)). A similar set of genes presumed to carryout the same function are encoded by CHY_(—)0731, CHY_(—)0732, andCHY_(—)0733 in C. hydrogenoformans (Wu et al., PLoS Genet. 1:e65(2005)). Homologs are also found in C. carboxidivorans P7.

TABLE 38 Protein GenBank ID GI Number Organism Moth_2312 YP_431142148283121 Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum_2703 YP_846816.1 116750129 Syntrophobacterfumaroxidans Sfum_2704 YP_846817.1 116750130 Syntrophobacterfumaroxidans Sfum_2705 YP_846818.1 116750131 Syntrophobacterfumaroxidans Sfum_2706 YP_846819.1 116750132 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY_0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY_0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7

Formyltetrahydrofolate synthetase ligates formate to tetrahydrofolate atthe expense of one ATP. This reaction is catalyzed by the gene productof Moth_(—)0109 in M. thermoacetica (Lovell et al., Arch. Microbiol.149:280-285 (1988); Lovell et al., Biochemistry 29:5687-5694 (1990);O'brien et al., Experientia. Suppl. 26:249-262 (1976), FHS inClostridium acidurici (Whitehead and Rabinowitz, J. Bacteriol.167:205-209 (1986); Whitehead and Rabinowitz, J. Bacteriol.170:3255-3261 (1988)), and CHY_(—)2385 in C. hydrogenoformans (Wu etal., PLoS Genet. 1:e65 (2005)). Homologs exist in C. carboxidivorans P7.

TABLE 39 Protein GenBank ID GI Number Organism Moth_0109 YP_428991.183588982 Moorella thermoacetica CHY_2385 YP_361182.1 78045024Carboxydothermus hydrogenoformans FHS P13419.1 120562 Clostridiumacidurici CcarbDRAFT_1913 ZP_05391913.1 255524966 Clostridiumcarboxidivorans P7 CcarbDRAFT_2946 ZP_05392946.1 255526022 Clostridiumcarboxidivorans P7

In M. thermoacetica, E. coli, and C. hydrogenoformans,methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolatedehydrogenase are carried out by the bi-functional gene products ofMoth_(—)1516, folD, and CHY_(—)1878, respectively (D'Ari and Rabinowitz,J. Biol. Chem. 266:23953-23958 (1991); Pierce et al., Environ.Microbiol. (2008); Wu et al., PLoS Genet. 1:e65 (2005)). A homologexists in C. carboxidivorans P7.

TABLE 40 Protein GenBank ID GI Number Organism Moth_1516 YP_430368.183590359 Moorella thermoacetica folD NP_415062.1 16128513 Escherichiacoli CHY_1878 YP_360698.1 78044829 Carboxydothermus hydrogenoformansCcarbDRAFT_2948 ZP_05392948.1 255526024 Clostridium carboxidivorans P7

The final step of the methyl branch of the Wood-Ljungdahl pathway iscatalyzed by methylenetetrahydrofolate reductase. In M. thermoacetica,this enzyme is oxygen-sensitive and contains an iron-sulfur cluster(Clark and Ljungdahl, J. Biol. Chem. 259:10845-10849 (1984)). Thisenzyme is encoded by metF in E. coli (Sheppard et al., J. Bacteriol.181:718-725 (1999)) and CHY_(—)1233 in C. hydrogenoformans (Wu et al.,PLoS Genet. 1:e65 (2005)). The M. thermoacetica genes, and its C.hydrogenoformans counterpart, are located near the CODH/ACS genecluster, separated by putative hydrogenase and heterodisulfide reductasegenes.

TABLE 41 Protein GenBank ID GI Number Organism metF NP_418376.1 16131779Escherichia coli CHY_1233 YP_360071.1 78044792 Carboxydothermushydrogenoformans

While E. coli naturally possesses the capability for some of therequired transformations (i.e., methenyltetrahydrofolate cyclohydrolase,methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolatereductase), it is thought that the methyl branch enzymes from acetogensmay have significantly higher (50-100×) specific activities than thosefrom non-acetogens (Morton et al., Genetics and molecular biology ofanaerobic bacteria, p. 389-406, Springer Verlag, New York (1992.)). Theformate dehydrogenase also appears to be specialized for anaerobicconditions (Ljungdahl and Andreesen, FEBS Lett. 54:279-282 (1975)).Therefore, various non-native versions of each of these are expressed inthe strain of E. coli capable of methanol and syngas utilization.Specifically, these genes are cloned and combined into an expressionvector designed to express them as a set. Initially, a high or mediumcopy number vector is chosen (using ColE1 or P15A replicons). Onepromoter that can be used is a strongly constitutive promoter such aslambda pL or an IPTG-inducible version of this, pL-lacO (Lutz andBujard, Nucleic Acids Res. 25:1203-1210 (1997)). To make an artificialoperon, one 5′ terminal promoter is placed upstream of the set of genesand each gene receives a consensus rbs element. The order of genes isbased on the natural order whenever possible. Ultimately, the genes areintegrated into the E. coli chromosome. Enzyme assays are performed asdescribed in (Clark and Ljungdahl, J. Biol. Chem. 259:10845-10849(1984); Clark and Ljungdahl, Meth. Enzymol. 122:392-399 (1986); D'Ariand Rabinowitz, J. Biol. Chem. 266:23953-23958 (1991); de Mata andRabinowitz, J. Biol. Chem. 255:2569-2577 (1980); Ljungdahl andAndreesen, Meth. Enzymol. 53:360-372 (1978); Lovell et al., 1988 Arch.Microbiol 149:280-285 (1988); Yamamoto et al., J. Biol. Chem.258:1826-1832 (1983)).

After strains of E. coli expressing both the carbonyl and methylbranches of the Wood-Ljungdahl pathway are constructed, they are assayedfor the ability to utilize syngas consisting of CO, CO₂, and/or H₂, forincorporation into acetyl-CoA, cell mass, and isopropanol or1,4-butanediol. Initial conditions employ strictly anaerobically growncells provided with exogenous glucose as a carbon and energy source.Metabolizing glucose or other carbohydrates to acetyl-CoA provides onepotential source of CO₂ that can be fixed via the Wood-Ljungdahlpathway. Alternatively, or in addition to glucose, nitrate will be addedto the fermentation broth to serve as an electron acceptor and initiatorof growth. Anaerobic growth of E. coli on fatty acids, which areultimately metabolized to acetyl-CoA, has been demonstrated in thepresence of nitrate (Campbell et al., Mol. Microbiol. 47:793-805(2003)). Oxygen can also be provided as long as its intracellular levelsare maintained below any inhibition threshold of the engineered enzymes.‘Synthetic syngas’ of a composition suitable for these experiments canalso be employed. ¹³C-labeled CO and/or CO₂ are provided to the cellsand analytical mass spectrometry is employed to measure incorporation ofthe labeled carbon into acetate, isopropanol, 4-hydroxybutyrate,1,4-butanediol, and cell mass (e.g., proteinogenic amino acids).

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing, or a protein associated with, thereferenced metabolic reaction, reactant or product. Unless otherwiseexpressly stated herein, those skilled in the art will understand thatreference to a reaction also constitutes reference to the reactants andproducts of the reaction. Similarly, unless otherwise expressly statedherein, reference to a reactant or product also references the reaction,and reference to any of these metabolic constituents also references thegene or genes encoding the enzymes that catalyze or proteins involved inthe referenced reaction, reactant or product. Likewise, given the wellknown fields of metabolic biochemistry, enzymology and genomics,reference herein to a gene or encoding nucleic acid also constitutes areference to the corresponding encoded enzyme and the reaction itcatalyzes or a protein associated with the reaction as well as thereactants and products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes or proteins participating in one or more isopropanol,4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathways. Depending onthe host microbial organism chosen for biosynthesis, nucleic acids forsome or all of a particular isopropanol, 4-hydroxybutyrate, or1,4-butanediol biosynthetic pathway can be expressed. For example, if achosen host is deficient in one or more enzymes or proteins for adesired biosynthetic pathway, then expressible nucleic acids for thedeficient enzyme(s) or protein(s) are introduced into the host forsubsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) or protein(s) to achieve isopropanol, 4-hydroxybutyrate, or1,4-butanediol biosynthesis. Thus, a non-naturally occurring microbialorganism of the invention can be produced by introducing exogenousenzyme or protein activities to obtain a desired biosynthetic pathway ora desired biosynthetic pathway can be obtained by introducing one ormore exogenous enzyme or protein activities that, together with one ormore endogenous enzymes or proteins, produces a desired product such asisopropanol, 4-hydroxybutyrate, or 1,4-butanediol.

Depending on the isopropanol, 4-hydroxybutyrate, or 1,4-butanediolbiosynthetic pathway constituents of a selected host microbial organism,the non-naturally occurring microbial organisms of the invention willinclude at least one exogenously expressed isopropanol,4-hydroxybutyrate, or 1,4-butanediol pathway-encoding nucleic acid andup to all encoding nucleic acids for one or more isopropanol,4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathways. For example,isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthesis can beestablished in a host deficient in a pathway enzyme or protein throughexogenous expression of the corresponding encoding nucleic acid. In ahost deficient in all enzymes or proteins of an isopropanol,4-hydroxybutyrate, or 1,4-butanediol pathway, exogenous expression ofall enzyme or proteins in the pathway can be included, although it isunderstood that all enzymes or proteins of a pathway can be expressedeven if the host contains at least one of the pathway enzymes orproteins. For example, exogenous expression of all enzymes or proteinsin a pathway for production of isopropanol, 4-hydroxybutyrate, or1,4-butanediol can be included.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel theisopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway deficienciesof the selected host microbial organism. Therefore, a non-naturallyoccurring microbial organism of the invention can have one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, up to all nucleic acids encoding the enzymesor proteins constituting an isopropanol, 4-hydroxybutyrate, or1,4-butanediol biosynthetic pathway disclosed herein. In someembodiments, the non-naturally occurring microbial organisms also caninclude other genetic modifications that facilitate or optimizeisopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthesis or thatconfer other useful functions onto the host microbial organism. One suchother functionality can include, for example, augmentation of thesynthesis of one or more of the isopropanol, 4-hydroxybutyrate, or1,4-butanediol pathway precursors such as acetyl-CoA.

Generally, a host microbial organism is selected such that it producesthe precursor of an isopropanol, 4-hydroxybutyrate, or 1,4-butanediolpathway, either as a naturally produced molecule or as an engineeredproduct that either provides de novo production of a desired precursoror increased production of a precursor naturally produced by the hostmicrobial organism. For example, acetyl-CoA is produced naturally in ahost organism such as E. coli. A host organism can be engineered toincrease production of a precursor, as disclosed herein. In addition, amicrobial organism that has been engineered to produce a desiredprecursor can be used as a host organism and further engineered toexpress enzymes or proteins of an isopropanol, 4-hydroxybutyrate, or1,4-butanediol pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize isopropanol, 4-hydroxybutyrate, or1,4-butanediol. In this specific embodiment it can be useful to increasethe synthesis or accumulation of an isopropanol, 4-hydroxybutyrate, or1,4-butanediol pathway product to, for example, drive isopropanol,4-hydroxybutyrate, or 1,4-butanediol pathway reactions towardisopropanol, 4-hydroxybutyrate, or 1,4-butanediol production. Increasedsynthesis or accumulation can be accomplished by, for example,overexpression of nucleic acids encoding one or more of theabove-described isopropanol, 4-hydroxybutyrate, or 1,4-butanediolpathway enzymes or proteins. Over expression the enzyme or enzymesand/or protein or proteins of the isopropanol, 4-hydroxybutyrate, or1,4-butanediol pathway can occur, for example, through exogenousexpression of the endogenous gene or genes, or through exogenousexpression of the heterologous gene or genes. Therefore, naturallyoccurring organisms can be readily generated to be non-naturallyoccurring microbial organisms of the invention, for example, producingisopropanol, 4-hydroxybutyrate, or 1,4-butanediol, throughoverexpression of one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, that is, upto all nucleic acids encoding isopropanol, 4-hydroxybutyrate, or1,4-butanediol biosynthetic pathway enzymes or proteins. In addition, anon-naturally occurring organism can be generated by mutagenesis of anendogenous gene that results in an increase in activity of an enzyme inthe isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosyntheticpathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, an isopropanol, 4-hydroxybutyrate, or 1,4-butanediolbiosynthetic pathway onto the microbial organism. Alternatively,encoding nucleic acids can be introduced to produce an intermediatemicrobial organism having the biosynthetic capability to catalyze someof the required reactions to confer isopropanol, 4-hydroxybutyrate, or1,4-butanediol biosynthetic capability. For example, a non-naturallyoccurring microbial organism having an isopropanol, 4-hydroxybutyrate,or 1,4-butanediol biosynthetic pathway can comprise at least twoexogenous nucleic acids encoding desired enzymes or proteins. Thus, itis understood that any combination of two or more enzymes or proteins ofa biosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes or proteins of a biosyntheticpathway can be included in a non-naturally occurring microbial organismof the invention and so forth, as desired, so long as the combination ofenzymes and/or proteins of the desired biosynthetic pathway results inproduction of the corresponding desired product. Similarly, anycombination of four, or more enzymes or proteins of a biosyntheticpathway as disclosed herein can be included in a non-naturally occurringmicrobial organism of the invention, as desired, so long as thecombination of enzymes and/or proteins of the desired biosyntheticpathway results in production of the corresponding desired product.

In addition to the biosynthesis of isopropanol, 4-hydroxybutyrate, or1,4-butanediol as described herein, the non-naturally occurringmicrobial organisms and methods of the invention also can be utilized invarious combinations with each other and with other microbial organismsand methods well known in the art to achieve product biosynthesis byother routes. For example, one alternative to produce isopropanol,4-hydroxybutyrate, or 1,4-butanediol other than use of the isopropanol,4-hydroxybutyrate, or 1,4-butanediol producers is through addition ofanother microbial organism capable of converting an isopropanol,4-hydroxybutyrate, or 1,4-butanediol pathway intermediate toisopropanol, 4-hydroxybutyrate, or 1,4-butanediol. One such procedureincludes, for example, the fermentation of a microbial organism thatproduces an isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathwayintermediate. The isopropanol, 4-hydroxybutyrate, or 1,4-butanediolpathway intermediate can then be used as a substrate for a secondmicrobial organism that converts the isopropanol, 4-hydroxybutyrate, or1,4-butanediol pathway intermediate to isopropanol, 4-hydroxybutyrate,or 1,4-butanediol. The isopropanol, 4-hydroxybutyrate, or 1,4-butanediolpathway intermediate can be added directly to another culture of thesecond organism or the original culture of the isopropanol,4-hydroxybutyrate, or 1,4-butanediol pathway intermediate producers canbe depleted of these microbial organisms by, for example, cellseparation, and then subsequent addition of the second organism to thefermentation broth can be utilized to produce the final product withoutintermediate purification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, isopropanol,4-hydroxybutyrate, or 1,4-butanediol. In these embodiments, biosyntheticpathways for a desired product of the invention can be segregated intodifferent microbial organisms, and the different microbial organisms canbe co-cultured to produce the final product. In such a biosyntheticscheme, the product of one microbial organism is the substrate for asecond microbial organism until the final product is synthesized. Forexample, the biosynthesis of isopropanol, 4-hydroxybutyrate, or1,4-butanediol can be accomplished by constructing a microbial organismthat contains biosynthetic pathways for conversion of one pathwayintermediate to another pathway intermediate or the product.Alternatively, isopropanol, 4-hydroxybutyrate, or 1,4-butanediol alsocan be biosynthetically produced from microbial organisms throughco-culture or co-fermentation using two organisms in the same vessel,where the first microbial organism produces an isopropanol,4-hydroxybutyrate, or 1,4-butanediol intermediate and the secondmicrobial organism converts the intermediate to isopropanol,4-hydroxybutyrate, or 1,4-butanediol.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce isopropanol,4-hydroxybutyrate, or 1,4-butanediol.

Sources of encoding nucleic acids for an isopropanol, 4-hydroxybutyrate,or 1,4-butanediol pathway enzyme or protein can include, for example,any species where the encoded gene product is capable of catalyzing thereferenced reaction. Such species include both prokaryotic andeukaryotic organisms including, but not limited to, bacteria, includingarchaea and eubacteria, and eukaryotes, including yeast, plant, insect,animal, and mammal, including human. Exemplary species for such sourcesinclude, for example, Escherichia coli, as well as other exemplaryspecies disclosed herein or available as source organisms forcorresponding genes. However, with the complete genome sequenceavailable for now more than 550 species (with more than half of theseavailable on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisiteisopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic activityfor one or more genes in related or distant species, including forexample, homologues, orthologs, paralogs and nonorthologous genedisplacements of known genes, and the interchange of genetic alterationsbetween organisms is routine and well known in the art. Accordingly, themetabolic alterations enabling biosynthesis of isopropanol,4-hydroxybutyrate, or 1,4-butanediol described herein with reference toa particular organism such as E. coli can be readily applied to othermicroorganisms, including prokaryotic and eukaryotic organisms alike.Given the teachings and guidance provided herein, those skilled in theart will know that a metabolic alteration exemplified in one organismcan be applied equally to other organisms.

In some instances, such as when an alternative isopropanol,4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathway exists in anunrelated species, isopropanol, 4-hydroxybutyrate, or 1,4-butanediolbiosynthesis can be conferred onto the host species by, for example,exogenous expression of a paralog or paralogs from the unrelated speciesthat catalyzes a similar, yet non-identical metabolic reaction toreplace the referenced reaction. Because certain differences amongmetabolic networks exist between different organisms, those skilled inthe art will understand that the actual gene usage between differentorganisms may differ. However, given the teachings and guidance providedherein, those skilled in the art also will understand that the teachingsand methods of the invention can be applied to all microbial organismsusing the cognate metabolic alterations to those exemplified herein toconstruct a microbial organism in a species of interest that willsynthesize isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus, algae, cyanobacteria or any of a variety of othermicroorganisms applicable to fermentation processes. Exemplary bacteriainclude species selected from Escherichia coli, Klebsiella oxytoca,Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis,Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor,Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonasputida. Exemplary yeasts or fungi include species selected fromSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus nigerand Pichia pastoris. Exemplary cyanobacteria include Acaryochlorismarina MBIC11017, Anabaena sp. PCC 7120, Anabaena variabilis ATCC 29413,Agmenellum quadruplicatum, Chlorobium tepidum TLS, Cyanothece sp. ATCC51142, Gloeobacter violaceus PCC 7421, Microcystis aeruginosa NIES-843,Nostoc punctiforme ATCC 29133, Prochlorococcus marinus MED4,Prochlorococcus marinus MIT9313, Prochlorococcus marinus SS120,Prochlorococcus marinus str. AS9601, Prochlorococcus marinus str. MIT9211, Prochlorococcus marinus str. MIT 9215, Prochlorococcus marinusstr. MIT 9301, Prochlorococcus marinus str. MIT 9303, Prochlorococcusmarinus str. MIT 9312, Prochlorococcus marinus str. MIT 9515,Prochlorococcus marinus str. NATL1A, Prochlorococcus marinus str.NATL2A, Rhodopseudomonas palustris CGA009, Synechococcus elongatus PCC6301, Synechococcus elongatus PCC 7942, Synechococcus sp. CC9311,Synechococcus sp. CC9605, Synechococcus sp. CC9902, Synechococcus sp.JA-2-3B\′a(2-13), Synechococcus sp. JA-3-3Ab, Synechococcus sp. PCC7002, Synechococcus sp. RCC307, Synechococcus sp. WH 7803, Synechococcussp. WH8102, Synechocystis sp. PCC 6803, Thermosynechococcus elongatusBP-1, Trichodesmium erythraeum IMS101. Exemplary algae includeBotryococcus braunii, Chlamydomonas reinhardii, Chlorella sp.,Crypthecodinium cohnii, Cylindrotheca sp., Dunaliella primolecta,Isochrysis sp., Monallanthus salina, Nannochloris sp., Nannochloropsissp., Neochloris oleoabundans, Nitzschia sp., Phaeodactylum tricornutum,Schizochytrium sp., and Tetraselmis sueica.

E. coli is a particularly useful host organism since it is a wellcharacterized microbial organism suitable for genetic engineering. Otherparticularly useful host organisms include yeast such as Saccharomycescerevisiae. It is understood that any suitable microbial host organismcan be used to introduce metabolic and/or genetic modifications toproduce a desired product.

Methods for constructing and testing the expression levels of anon-naturally occurring isopropanol, 4-hydroxybutyrate, or1,4-butanediol-producing host can be performed, for example, byrecombinant and detection methods well known in the art. Such methodscan be found described in, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory,New York (2001); and Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofisopropanol, 4-hydroxybutyrate, or 1,4-butanediol can be introducedstably or transiently into a host cell using techniques well known inthe art including, but not limited to, conjugation, electroporation,chemical transformation, transduction, transfection, and ultrasoundtransformation. For exogenous expression in E. coli or other prokaryoticcells, some nucleic acid sequences in the genes or cDNAs of eukaryoticnucleic acids can encode targeting signals such as an N-terminalmitochondrial or other targeting signal, which can be removed beforetransformation into prokaryotic host cells, if desired. For example,removal of a mitochondrial leader sequence led to increased expressionin E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)).For exogenous expression in yeast or other eukaryotic cells, genes canbe expressed in the cytosol without the addition of leader sequence, orcan be targeted to mitochondrion or other organelles, or targeted forsecretion, by the addition of a suitable targeting sequence such as amitochondrial targeting or secretion signal suitable for the host cells.Thus, it is understood that appropriate modifications to a nucleic acidsequence to remove or include a targeting sequence can be incorporatedinto an exogenous nucleic acid sequence to impart desirable properties.Furthermore, genes can be subjected to codon optimization withtechniques well known in the art to achieve optimized expression of theproteins.

An expression vector or vectors can be constructed to include one ormore isopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosyntheticpathway encoding nucleic acids as exemplified herein operably linked toexpression control sequences functional in the host organism. Expressionvectors applicable for use in the microbial host organisms of theinvention include, for example, plasmids, phage vectors, viral vectors,episomes and artificial chromosomes, including vectors and selectionsequences or markers operable for stable integration into a hostchromosome. Additionally, the expression vectors can include one or moreselectable marker genes and appropriate expression control sequences.Selectable marker genes also can be included that, for example, provideresistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more exogenousencoding nucleic acids are to be co-expressed, both nucleic acids can beinserted, for example, into a single expression vector or in separateexpression vectors. For single vector expression, the encoding nucleicacids can be operationally linked to one common expression controlsequence or linked to different expression control sequences, such asone inducible promoter and one constitutive promoter. The transformationof exogenous nucleic acid sequences involved in a metabolic or syntheticpathway can be confirmed using methods well known in the art. Suchmethods include, for example, nucleic acid analysis such as Northernblots or polymerase chain reaction (PCR) amplification of mRNA, orimmunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the exogenous nucleic acid is expressed in asufficient amount to produce the desired product, and it is furtherunderstood that expression levels can be optimized to obtain sufficientexpression using methods well known in the art and as disclosed herein.

The invention provides a method for producing isopropanol that includesculturing a non-naturally occurring microbial organism having anisopropanol pathway. The pathway includes at least one exogenous nucleicacid encoding an isopropanol pathway enzyme expressed in a sufficientamount to produce isopropanol under conditions and for a sufficientperiod of time to produce isopropanol. The isopropanol pathwaycomprising an acetoacetyl-CoA thiolase, an acetoacetyl-CoA transferase,an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, aphosphotransacetoacetylase, an acetoacetate kinase, an acetoacetatedecarboxylase, and an isopropanol dehydrogenase. An alternativeisopropanol pathway comprises acetoacetyl-CoA thiolase,succinyl-CoA:3-ketoacid CoA transferase, acetoacetate decarboxylase, andan isopropanol dehydrogenase.

In embodiments where an organism has a methanol methyltransferase,culturing can be carried out utilizing a feedstock such as 1) methanoland CO, 2) methanol, CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4)methanol and synthesis gas comprising CO and H₂, 5) methanol andsynthesis gas comprising CO, CO₂, and H₂, 6) one or more carbohydrates,7) methanol and one or more carbohyrates, and 8) methanol. Exemplarycarbohydrates include but are not limited to glucose, sucrose, xylose,arabinose and glycerol.

In embodiments where an organism has a formate dehydrogenase, aformyltetrahydrofolate synthetase, a methenyltetrahydrofolatecyclohydrolase, a methylenetetrahydrofolate dehydrogenase, and amethylenetetrahydrofolate reductase, the organism can utilize afeedstock such as 1) CO, 2) CO₂ and H₂, 3) CO and CO₂, 4) synthesis gascomprising CO and H₂, 5) synthesis gas comprising CO, CO₂, and H₂, and6) one or more carbohydrates.

Similarly, 4-hydroxybutyrate or 1,4-butanediol can also be produced byculturing the appropriate organisms as described herein above.

Suitable purification and/or assays to test for the production ofisopropanol, 4-hydroxybutyrate, or 1,4-butanediol can be performed usingwell known methods. Suitable replicates such as triplicate cultures canbe grown for each engineered strain to be tested. For example, productand byproduct formation in the engineered production host can bemonitored. The final product and intermediates, and other organiccompounds, can be analyzed by methods such as HPLC (High PerformanceLiquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) andLC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitableanalytical methods using routine procedures well known in the art. Therelease of product in the fermentation broth can also be tested with theculture supernatant. Byproducts and residual glucose can be quantifiedby HPLC using, for example, a refractive index detector for glucose andalcohols, and a UV detector for organic acids (Lin et al., Biotechnol.Bioeng. 90:775-779 (2005)), or other suitable assay and detectionmethods well known in the art. The individual enzyme or proteinactivities from the exogenous DNA sequences can also be assayed usingmethods well known in the art (see, for example, WO/2008/115840 andHanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007)).

The isopropanol, 4-hydroxybutyrate, or 1,4-butanediol can be separatedfrom other components in the culture using a variety of methods wellknown in the art. Such separation methods include, for example,extraction procedures as well as methods that include continuousliquid-liquid extraction, pervaporation, membrane filtration, membraneseparation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the isopropanol, 4-hydroxybutyrate, or1,4-butanediol producers can be cultured for the biosynthetic productionof isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.

For the production of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol,the recombinant strains are cultured in a medium with carbon source andother essential nutrients. It is highly desirable to maintain anaerobicconditions in the fermenter to reduce the cost of the overall process.Such conditions can be obtained, for example, by first sparging themedium with nitrogen and then sealing the flasks with a septum andcrimp-cap. For strains where growth is not observed anaerobically,microaerobic conditions can be applied by perforating the septum with asmall hole for limited aeration. Exemplary anaerobic conditions havebeen described previously and are well-known in the art. Exemplaryaerobic and anaerobic conditions are described, for example, in U.S.patent application Ser. No. 11/891,602, filed Aug. 10, 2007.Fermentations can be performed in a batch, fed-batch or continuousmanner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

In addition to renewable feedstocks such as those exemplified above, theisopropanol, 4-hydroxybutyrate, or 1,4-butanediol microbial organisms ofthe invention also can be modified for growth on syngas as its source ofcarbon. In this specific embodiment, one or more proteins or enzymes areexpressed in the isopropanol, 4-hydroxybutyrate, or 1,4-butanediolproducing organisms to provide a metabolic pathway for utilization ofsyngas or other gaseous carbon source.

Although organisms of the present invention are designed to utilizesyngas and/or methanol as a growth source, they may also utilize, forexample, any carbohydrate source which can supply a source of carbon tothe non-naturally occurring microorganism. Such sources include, forexample, sugars such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Other sources of carbohydrate include, for example,renewable feedstocks and biomass. Exemplary types of biomasses that canbe used as feedstocks in the methods of the invention include cellulosicbiomass, hemicellulosic biomass and lignin feedstocks or portions offeedstocks. Such biomass feedstocks contain, for example, carbohydratesubstrates useful as carbon sources such as glucose, xylose, arabinose,galactose, mannose, fructose and starch. Given the teachings andguidance provided herein, those skilled in the art will understand thatrenewable feedstocks and biomass other than those exemplified above alsocan be used for culturing the microbial organisms of the invention forthe production of isopropanol, 4-hydroxybutyrate, or 1,4-butanediol.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as syngas,methanol, or combinations of CO, CO₂, hydrogen, and the like. Suchcompounds include, for example, isopropanol, 4-hydroxybutyrate, or1,4-butanediol and any of the intermediate metabolites in theisopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway. All that isrequired is to engineer in one or more of the required enzyme or proteinactivities to achieve biosynthesis of the desired compound orintermediate including, for example, inclusion of some or all of theisopropanol, 4-hydroxybutyrate, or 1,4-butanediol biosynthetic pathways.Accordingly, the invention provides a non-naturally occurring microbialorganism that produces and/or secretes isopropanol, 4-hydroxybutyrate,or 1,4-butanediol when grown on a carbohydrate or other carbon sourceand produces and/or secretes any of the intermediate metabolites shownin the isopropanol, 4-hydroxybutyrate, or 1,4-butanediol pathway whengrown on a carbohydrate or other carbon source. The isopropanol,4-hydroxybutyrate, or 1,4-butanediol producing microbial organisms ofthe invention can initiate synthesis from an intermediate, for example,acetyl-CoA.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding an isopropanol,4-hydroxybutyrate, or 1,4-butanediol pathway enzyme or protein insufficient amounts to produce isopropanol, 4-hydroxybutyrate, or1,4-butanediol. It is understood that the microbial organisms of theinvention are cultured under conditions sufficient to produceisopropanol, 4-hydroxybutyrate, or 1,4-butanediol. Following theteachings and guidance provided herein, the non-naturally occurringmicrobial organisms of the invention can achieve biosynthesis ofisopropanol, 4-hydroxybutyrate, or 1,4-butanediol resulting inintracellular concentrations between about 0.1-2000 mM or more.Generally, the intracellular concentration of isopropanol,4-hydroxybutyrate, or 1,4-butanediol is between about 3-1800 mM,particularly between about 5-1700 mM and more particularly between about8-1600 mM, including about 100 mM, 200 mM, 500 mM, 800 mM, or more.Intracellular concentrations between and above each of these exemplaryranges also can be achieved from the non-naturally occurring microbialorganisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007. Any of theseconditions can be employed with the non-naturally occurring microbialorganisms as well as other anaerobic conditions well known in the art.Under such anaerobic conditions, the isopropanol, 4-hydroxybutyrate, or1,4-butanediol producers can synthesize isopropanol, 4-hydroxybutyrate,or 1,4-butanediol at intracellular concentrations of 5-10 mM or more aswell as all other concentrations exemplified herein. It is understoodthat, even though the above description refers to intracellularconcentrations, isopropanol, 4-hydroxybutyrate, or 1,4-butanediolproducing microbial organisms can produce isopropanol,4-hydroxybutyrate, or 1,4-butanediol intracellularly and/or secrete theproduct into the culture medium.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of isopropanol, 4-hydroxybutyrate, or 1,4-butanediolincludes anaerobic culture or fermentation conditions. In certainembodiments, the non-naturally occurring microbial organisms of theinvention can be sustained, cultured or fermented under anaerobic orsubstantially anaerobic conditions. Briefly, anaerobic conditions refersto an environment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of isopropanol, 4-hydroxybutyrate, or1,4-butanediol. Exemplary growth procedures include, for example,fed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation. All of these processes are well known in the art.Fermentation procedures are particularly useful for the biosyntheticproduction of commercial quantities of isopropanol, 4-hydroxybutyrate,or 1,4-butanediol. Generally, and as with non-continuous cultureprocedures, the continuous and/or near-continuous production ofisopropanol, 4-hydroxybutyrate, or 1,4-butanediol will include culturinga non-naturally occurring isopropanol, 4-hydroxybutyrate, or1,4-butanediol producing organism of the invention in sufficientnutrients and medium to sustain and/or nearly sustain growth in anexponential phase. Continuous culture under such conditions can beinclude, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 ormore weeks and up to several months. Alternatively, organisms of theinvention can be cultured for hours, if suitable for a particularapplication. It is to be understood that the continuous and/ornear-continuous culture conditions also can include all time intervalsin between these exemplary periods. It is further understood that thetime of culturing the microbial organism of the invention is for asufficient period of time to produce a sufficient amount of product fora desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of isopropanol, 4-hydroxybutyrate, or1,4-butanediol can be utilized in, for example, fed-batch fermentationand batch separation; fed-batch fermentation and continuous separation,or continuous fermentation and continuous separation. Examples of batchand continuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the isopropanol,4-hydroxybutyrate, or 1,4-butanediol producers of the invention forcontinuous production of substantial quantities of isopropanol,4-hydroxybutyrate, or 1,4-butanediol, the isopropanol,4-hydroxybutyrate, or 1,4-butanediol producers also can be, for example,simultaneously subjected to chemical synthesis procedures to convert theproduct to other compounds or the product can be separated from thefermentation culture and sequentially subjected to chemical conversionto convert the product to other compounds, if desired.

Important process considerations for a syngas fermentation are highbiomass concentration and good gas-liquid mass transfer (Bredwell etal., Biotechnol. Prog. 15:834-844 (1999). The solubility of CO in wateris somewhat less than that of oxygen. Continuously gas-spargedfermentations can be performed in controlled fermenters with constantoff-gas analysis by mass spectrometry and periodic liquid sampling andanalysis by GC and HPLC. The liquid phase can function in batch mode.Fermentation products such as alcohols, organic acids, and residualglucose along with residual methanol are quantified by HPLC (Shimadzu,Columbia Md.), for example, using an Aminex® series of HPLC columns (forexample, HPX-87 series) (BioRad, Hercules Calif.), using a refractiveindex detector for glucose and alcohols, and a UV detector for organicacids. The growth rate is determined by measuring optical density usinga spectrophotometer (600 nm). All piping in these systems is glass ormetal to maintain anaerobic conditions. The gas sparging is performedwith glass frits to decrease bubble size and improve mass transfer.Various sparging rates are tested, ranging from about 0.1 to 1 vvm(vapor volumes per minute). To obtain accurate measurements of gasuptake rates, periodic challenges are performed in which the gas flow istemporarily stopped, and the gas phase composition is monitored as afunction of time.

In order to achieve the overall target productivity, methods of cellretention or recycle are employed. One method to increase the microbialconcentration is to recycle cells via a tangential flow membrane from asidestream. Repeated batch culture can also be used, as previouslydescribed for production of acetate by Moorella (Sakai et al., J.Biosci. Bioeng. 99:252-258 (2005)). Various other methods can also beused (Bredwell et al., Biotechnol. Prog. 15:834-844 (1999); Datar etal., Biotechnol. Bioeng. 86:587-594 (2004)). Additional optimization canbe tested such as overpressure at 1.5 atm to improve mass transfer(Najafpour and Younesi, Enzyme Microb. Technol. 38[1-2], 223-228(2006)).

Once satisfactory performance is achieved using pure H₂/CO as the feed,synthetic gas mixtures are generated containing inhibitors likely to bepresent in commercial syngas. For example, a typical impurity profile is4.5% CH₄, 0.1% C₂H₂, 0.35% C₂H₆, 1.4% C₂H₄, and 150 ppm nitric oxide(Datar et al., Biotechnol. Bioeng. 86:587-594 (2004)). Tars, representedby compounds such as benzene, toluene, ethylbenzene, p-xylene, o-xylene,and naphthalene, are added at ppm levels to test for any effect onproduction. For example, it has been shown that 40 ppm NO is inhibitoryto C. carboxidivorans (Ahmed and Lewis, Biotechnol. Bioeng. 97:1080-1086(2007)). Cultures are tested in shake-flask cultures before moving to afermentor. Also, different levels of these potential inhibitorycompounds are tested to quantify the effect they have on cell growth.This knowledge is used to develop specifications for syngas purity,which is utilized for scale up studies and production. If any particularcomponent is found to be difficult to decrease or remove from syngasused for scale up, an adaptive evolution procedure is utilized to adaptcells to tolerate one or more impurities.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of isopropanol,4-hydroxybutyrate, or 1,4-butanediol.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion strategies that result in genetically stablemicroorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microorganism in order to suggest genetic manipulations that force thedesired biochemical to become an obligatory byproduct of cell growth. Bycoupling biochemical production with cell growth through strategicallyplaced gene deletions or other functional gene disruption, the growthselection pressures imposed on the engineered strains after long periodsof time in a bioreactor lead to improvements in performance as a resultof the compulsory growth-coupled biochemical production. Lastly, whengene deletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I ACS/CODH Gene Insertions in E. coli

This example describes the creation of E. coli plasmids that express theM. thermoacetica ACS/CODH operon genes including those required forCODH, ACS, methyltransferase, and the corrinoid iron-sulfur protein.This example further describes the expression these in E. coli resultingin observable CO oxidation activity, methyltransferase activity, andcorrinoid iron-sulfur protein activity. Finally, this exampledemonstrates that E. coli tolerates high CO concentrations, and may evenconsume CO when the CO-utilizing gene products from M. thermoacetica areexpressed.

Expression vectors were chosen from the set described by Lutz and Bujard(Lutz and Bujard, Nucleic Acids Res. 25:1203-1210 (1997)); these comewith compatible replicons that cover a range of copy numbers.Additionally, each contains prAl-lacol; this T7 early gene promoter isinducible by IPTG and can lead to very high levels of transcription inthe presence of IPTG and represses in other conditions. TheACS/CODH-encoding operon was cloned from Moth_(—)1204 (cooC) toMoth_(—)1197; a second version containing only Moth_(—)1203 toMoth_(—)1197 was also constructed. Both of these fragments (10-11 kbp)were confirmed by DNA sequence analysis. These were constructed in bothp15A and ColE1-based vectors for medium to high copy numbers.

To estimate the final concentrations recombinant proteins, SDS-PAGEfollowed by Western blot analyses were performed on the same cellextracts used in the CO oxidation, ACS, methyltransferase, and corrinoidFe—S assays. The antisera used were polyclonal to purified M.thermoacetica ACS/CODH and Mtr proteins and were visualized using analkaline phosphatase-linked goat-anti-rabbit secondary antibody. TheWesterns Blots are shown in FIGS. 4A and 4B. Amounts of CODH in ACS90and ACS91 were estimated at 50 ng by comparison to the control lanes.

A carbon monoxide oxidation assay (Seravalli et al., Biochemistry43:3944-3955 (2004)) was used to test whether or not functionalexpression of the CODH-encoding genes from M. thermoacetica wasachieved. Cultures of E. coli MG1655 containing either an empty vector,or the vectors expressing “Acs90” or “Acs91” were grown in TerrificBroth under anaerobic conditions (with supplements of cyanocobalamin,ferrous iron, and reducing agents) until reaching medium to high densityat which point, IPTG was added to a final concentration of 0.2 mM toinduce the promoter. After 3.5 hrs of growth at 37 C, the cells wereharvested and spun down prior to lysis with lysozyme and milddetergents. There is a benchmark figure of M. thermoacetica CODHspecific activity, 500 U at 55 C or ˜60 U at 25 C. This assay employedreduction of methyl viologen in the presence of CO. This is measured at578 nm in stoppered, anaerobic, glass cuvettes. Reactions positive forCO oxidation by CODH turned a deep violet color (see FIG. 5). About 0.5%of the cellular protein was CODH as estimated by Western blotting;therefore, the data in Table 42 are approximately 50× less than the 500U/mg activity of pure M. thermoacetica CODH. Nevertheless, thisexperiment did clearly demonstrate CO oxidation activity in recombinantE. coli with a much smaller amount in the negative controls. The smallamount of CO oxidation (CH₃ viologen reduction) seen in the negativecontrols indicates that E. coli may have a limited ability to reduce CH₃viologen.

TABLE 42 ACS90 7.7 mg/ml ACS91 11.8 mg/ml Mta98 9.8 mg/ml Mta99 11.2mg/ml Extract Vol OD/ U/ml U/mg ACS90 10 microliters 0.073 0.376 0.049ACS91 10 microliters 0.096 0.494 0.042 Mta99 10 microliters 0.0031 0.0160.0014 ACS90 10 microliters 0.099 0.51 0.066 Mta99 25 microliters 0.0120.025 0.0022 ACS91 25 microliters 0.215 0.443 0.037 Mta98 25 microliters0.019 0.039 0.004 ACS91 10 microliters 0.129 0.66 0.056 Averages ACS900.057 U/mg ACS91 0.045 U/mg Mta99 0.0018 U/mg

This assay is an in vitro reaction that synthesizes acetyl-CoA frommethyl-tetrahydrofolate, CO, and CoA using ACS/CODH, methyltransferase,and CFeSP (Raybuck et al., Biochemistry 27:7698-7702 (1988)). By addingor leaving out each of the enzymes involved, this assay can be used fora wide range of experiments, from testing one or more purified enzymesor cell extracts for activity, to determining the kinetics of thereaction under various conditions or with limiting amounts of substrateor enzyme. Samples of the reaction taken at various time points arequenched with 1M HCl, which liberates acetate from the acetyl-CoA endproduct. After purification with Dowex columns, the acetate can beanalyzed by chromatography, mass spectrometry, or by measuringradioactivity. The exact method will be determined by the specificsubstrates used in the reaction.

This assay was run in order to determine if the ACS/CODH operonexpressed in E. coli expresses the Fe—S corrinoid protein activity.Therefore, 14C-labeled methyl-THF was used as a labeled substrate tomeasure acetate synthesis by radioactivity incorporation into isolatedacetate samples. Six different conditions were tested:

-   -   1. Purified ACS/CODH, MeTr, and CFeSP as a positive control    -   2. Purified ACS/CODH with ACS90 cell extract    -   3. Purified ACS/CODH with ACS91 cell extract    -   4. Purified ACS/CODH, MeTr with ACS90 cell extract    -   5. Purified ACS/CODH, MeTr with ACS91 cell extract    -   6. Purified ACS/CODH, MeTr with as much ACS91 cell extract as        possible (excluding the MES buffer)

The reaction was assembled in the anaerobic chamber in assay vialsfilled with CO. The total reaction volume was small compared to the vialvolume, reagents were added prior to filling with CO, a gas-tightHamilton syringe was used and the reagents were kept anaerobic. Thereaction (˜60 ul total) consisted of the cell extract (except #1), CoA,Ti(III) citrate, MES (except #6), purified ACS/CODH,14C-methyl-tetrahydrofolate, methyl-viologen, and ferredoxin.Additionally, purified MeTr was added to #1, #4-6 and purified CFeSP wasadded to #1.

The reaction was carried out in the anaerobic chamber in a sand bath at55°. The final reagent added was the 14C-methyl-tetrahydrofolate, whichstarted the reaction (t=0 s). An initial sample was taken immediately,followed by samples at 30 minutes, 1 hour, and 2 hours. These timepoints are not exact, as the 6 conditions were run concurrently (sincethis experiment was primarily a qualitative one). The 15 ul samples wereadded to 15 ul of 1M HCl in scintillation vials. After counting thereaction mixtures, it was determined that the corrinoid Fe—S protein inACS90 extracts was active with total activity approaching approximately⅕ of the positive control.

Within the ACS/CODH operon is encoded an essential methyltransferaseactivity that catalyzes the transfer of CH₃ from methyl-tetrahydrofolateto the ACS complex as part of the synthesis of acetyl-CoA (i.e. this isthe step that the methyl and carbonyl paths join together). Within theoperon in M. thermoacetica, the Mtr-encoding gene is Moth_(—)1197 andcomes after the main CODH and ACS subunits. Therefore, Mtr activitywould constitute indirect evidence that the more proximal genes can beexpressed.

Mtr activity was assayed by spectroscopy. Specifically, methylatedCFeSP, with Co(III), has a small absorption peak at ˜450 nm, whilenon-methylated CFeSP, with Co(I), has a large peak at ˜390 nm. Thisspectrum is due to both the cobalt and iron-sulfur cluster chromophores.Additionally, it should be noted that the CFeSP can spontaneouslyoxidize to Co(II), which creates a broad absorption peak at ˜470 nm(Seravalli et al., Biochem. 38:5728-5735 (1999)). See FIG. 6 for theresults from E. coli cells containing ACS90.

To test whether or not E. coli can grow anaerobically in the presence ofsaturating amounts of CO, 120 ml serum bottles with 50 ml of TerrificBroth medium (plus NiCl₂, Fe(II)NH₄SO₄, and cyanocobalamin) were made inanaerobic conditions. One half of these bottles were equilibrated withnitrogen gas for 30 min. and one half was equilibrated with CO gas for30 min. An empty vector (pZA33) was used as a control and that and bothACS90 and ACS91 were tested with both N₂ and CO. All were grown for 36hrs with shaking (250 rpm) at 37 C. At the end of the 36 period,examination of the flasks showed high amounts of growth in all (FIG. 7).The bulk of the observed growth occurred overnight with a long lag ofsome (low but visible) density. Inocula sizes were ˜0.5 ml from E. colistocks.

The final CO concentrations are measured using an assay of the spectralshift of myoglobin upon exposure to CO. Myoglobin reduced with sodiumdithionite has an absorbance peak at 435 nm; this peak is shifted to 423nm with CO. Due to the low wavelength (and need to record a wholespectrum from 300 nm on upwards) quartz cuvettes must be used. COconcentration is measured against a standard curve and depends upon theHenry's Law constant for CO of maximum water solubility=970 micromolarat 20 C and 1 atm.

The results shown in Table 43 are very encouraging. Growth reachedsimilar levels (by visual inspection) whether or not a strain wascultured in the presence of CO or not. Furthermore, the negative controlhad a final CO concentration of 930 micromolar vs. 688 and 728micromolar for the ACS/CODH operon expressing strains. Clearly, theerror in these measurements is high given the large standard deviations.Nevertheless, this test does allow two tentative conclusions: 1) E. colican tolerate exposure to CO under anaerobic conditions, and 2) E. colicells expressing the ACS/CODH operon might be metabolizing some of theCO. The second conclusion is significantly less certain than the first.

TABLE 43 Stain and Final CO Growth Conditions concentration pZA33-CO(micromolar) ACS90-CO 638 494 734 883 ave 687 SD 164 ACS91-CO 728 812760 611 ave. 728 SD 85

Example II Enhancing The Yield of Fermentation Products from Sugars withWood-Ljungdahl Pathway Enzymes

In this example, we describe a non-naturally occurring microorganismexpressing genes encoding enzymes that catalyze the carbonyl-branch ofthe Wood-Ljungdahl pathway. Wood-Ljungdahl pathway enzymes assimilatecarbon in the form of CO and/or CO₂ into acetyl-CoA, which cansubsequently be converted to useful chemical products such asisopropanol, 4-hydroxybutyrate (4-HB) and 1,4-butanediol (14-BDO). TheWood-Ljungdahl pathway can also serve as a secondary carbon assimilationpathway during growth on other substrates such as glucose. In thiscapacity, WL pathway enzymes harness reducing equivalents generated inglycolysis to convert CO₂ to acetyl-CoA, cell mass and downstreamproducts at high yield.

Isopropanol: Engineering a Wood-Ljungdahl pathway into aglucose-utilizing and isopropanol-producing organism improves themaximum isopropanol yield up to 33%. The production of isopropanol hasbeen demonstrated in E. coli (Nahvi et al., Chem. Biol. 9:1043 (2002)).Specifically, acetyl CoA can be converted into acetoacetyl CoA,transformed into acetoacetate, decarboxylated to form acetone and thenreduced to form isopropanol (see FIG. 1). This pathway isredox-imbalanced and yields only 1 mol IPA/mol glc or 0.333 g/g. If theIPA pathway is supplemented with CO₂-fixation by the Wood-Ljungdahlpathway, the theoretical stoichiometric yield of IPA (1.33 mol/mol,0.444 lb/lb) is achievable, the pathway is redox-balanced, and cangenerate energy.

FIG. 8A shows a flux distribution for the conversion of one mole ofglucose into 1.33 moles of isopropanol. The conversion of one mole ofglucose to two moles of pyruvate generates four reducing equivalents andtwo ATPs. Pyruvate is subsequently converted to molar quantities ofacetyl-CoA by one or more enzymes including pyruvate formate lyase(PFL), pyruvate dehydrogenase (PDH) or pyruvate ferredoxinoxidoreductase (PFOR). PDH and PFOR generate molar quantities of CO₂ andtwo reducing equivalents per pyruvate consumed. Pyruvate formate lyaseforms formate as a byproduct, which can be converted to CO₂ and tworeducing equivalents by formate dehydrogenase. In the absence of WLpathway enzymes, the two moles of acetyl-CoA are converted to one moleof isopropanol. Assuming reducing equivalents are generated from thepyruvate to acetyl-CoA conversion, this pathway generates an excess of 3reducing equivalents per glucose consumed. When an active WL pathway ispresent 0.66 moles of CO₂ or formate (per mole glucose consumed) areassimilated into acetyl-CoA, which is subsequently converted to 0.33moles of isopropanol. This combined glycolysis/Wood Ljungdahl pathway isexactly balanced from a redox standpoint and is also capable ofgenerating energy. Two ATPs are generated in the conversion of glucoseto pyruvate. In this example, an additional 1.33 ATPs are recovered fromconversion of acetyl-CoA to acetate, yielding a total supply of 3.33ATPs per glucose consumed. The Wood-Ljungdahl pathway consumes 0.67 ATPsleaving 2.67 ATPs available for cell growth and metabolism.

4-Hydroxybutyrate: A similar improvement in yield is achievable for theproduction of 4-hydroxybutyrate. The maximum 4-HB yield from glucosealone is 1 mole 4-HB per mole glucose consumed (0.578 g/g). The pathway,shown in FIG. 8B, is redox-imbalanced and requires aeration and/or theformation of fermentation byproducts. An organism with a WL pathway canachieve the maximum stoichiometric yield of 4-HB (1.33 mol/mol or 0.768g/g). A flux distribution of the maximum theoretical yield is shown inFIG. 8B. Like the isopropanol pathway described previously, the pathwayis redox-balanced and yields a maximum availability of 2.67 ATP for cellgrowth and maintenance.

1,4-Butanediol: 1,4-Butanediol can also be synthesized from acetyl-CoAvia the intermediate 4-hydroxybutyryl-CoA. In this pathway,acetoacetyl-CoA is converted to 4-hydroxybutyryl-CoA as describedpreviously in the 4-hydroxybutyrate pathway. 4-Hydroxybutyryl-CoA issubsequently converted to 14-BDO by a bifunctional aldehydedehydrogenase or a CoA-dependent aldehyde dehydrogenase and alcoholdehydrogenase. Alternatively, it can be converted to 4-hydroxybutyratewhich can be first reduced directly to 4-hydroxybutyraldehyde andsubsequently reduced to 1,4-butanediol. As this pathway is notredox-balaced, the maximum 14-BDO yield of this pathway from sugars is 1mol/mol (0.5 g/g). Additional assimilation of CO₂ via Wood-Ljungdahlpathway further improves the yield to the theoretical maximum of 1.09mol/mol (0.545 g/g). A predicted flux distribution for achieving themaximum theoretical yield is shown in FIG. 8C. In this pathway, 2 ATPsare generated in glycolysis and 0.18 ATPs are consumed in the WLpathway, leaving a maximum of 1.82 ATPs available for cell growth andmaintenance.

Example III Enhancing The Yield of Fermentation Products from Sugarswith Wood-Ljungdahl Pathway Enzymes and a MtaABC-type methyltransferasesystem

An organism engineered to express genes encoding an ACS/CODH enzyme anda MtaABC-type methyltransferase system is capable of producingacetyl-CoA and downstream products at high yields from glucose andmethanol. The methyltransferase system enables the assimilation ofmethanol into 5-methyl-tetrahydrofolate (Me-THF). The CODH/ACS enzymeconfers the functionality to convert CO₂ to CO and CO, CoA and Me-THF toacetyl-CoA. The recombinant organism can be further engineered totransform acetyl-CoA to products of interest such as isopropanol,4-hydroxybutyrate, or 1,4-butanediol. Microorganisms with these featuresare capable of metabolizing sugars in conjunction with methanol toachieve high yields of chemical products. The ratio of glucose tomethanol consumed impacts the overall product yield.

An organism engineered to produce isopropanol from glucose viaacetyl-CoA achieves a maximum isopropanol yield of 1 mole product permole glucose utilized. Co-feeding methanol to this organism at a ratioof 2 methanol per one glucose further increases the yield to a maximumof 2 moles isopropanol per mole of glucose consumed. In the maximumyield scenario 2 moles of methanol are utilized per glucose consumed,producing 2 moles of IPA. In addition, 2 moles of CO₂ are assimilatedinto acetyl-CoA via ACS/CODH. The reduction of CO₂ to CO by ACS/CODHconsumes reducing equivalents, enabling redox balance and improvedproduct yield. The energetic yield of this pathway is also favorable,with 4 ATP equivalents generated per glucose consumed. An exemplary fluxdistribution of the conversion of methanol and glucose to isopropanol isshown in FIG. 9. The relationship between the glucose/methanol feedratio and the maximum product yield in FIG. 10.

A similar improvement in yield is observed for a 4-HB-producing organismutilizing the 4-HB pathway from acetyl-CoA in FIG. 2. One mole ofglucose and two moles each of methanol and CO₂ are assimilated in aredox-balanced pathway to form two moles of 4-hydroxybutyrate.Similarly, the yield of 1,4-BDO from carbohydrates can be improved byco-utilizing methanol.

Example IV Engineering Cobalamin Synthesis into an Organism

The key enzyme of the Wood-Ljungdahl pathway, ACS/CODH, requirescobalamin (vitamin B₁₂) to function. B₁₂ is synthesized de novo in someorganisms but must be supplied exogenously to others. Still otherorganisms such as S. cerevisiae lack the ability to efficiently uptakeB₁₂. This example describes a strategy for engineering de novo B₁₂synthetic capability into an organism.

B₁₂ biosynthetic pathways have been characterized in several organismsincluding Salmonella typhimurium LT2 (Roth et al., J. Bacteriol.175:3303-3316 (1993)), Lactobacillus reuteri CRL1098 (Sennett et al.,Annu. Rev. Biochem. 50:1053-1086 (1981)) and Bacillus megaterium (Breyet al., J. Bacteriol. 167:623-630 (1986)). Bacterial B₁₂ biosynthesispathways involve 20-30 genes clustered together in one or more operons.Two cobalamin biosynthesis pathways: late-insertion (aerobic only) andearly-insertion (anaerobic) have been described (Scott, A. I., J. Org.Chem. 68:2529-2539 (2003)). The final products of the biosynthesis ofvitamin B₁₂ are 5′-deoxyadenosylcobalamin (coenzyme B₁₂) andmethylcobalamin (MeCbl). Vitamin B₁₂ is defined as cyanocobalamin(CNCbl) which is the form commonly prepared in industry. In thisexample, B₁₂ refers to all three analogous molecules.

The anaerobic cobalamin biosynthesis pathway has been well-characterizedin Salmonella typhimurium LT2 (Roth et al., J. Bacteriol. 175:3303-3316(1993)). Pathway genes are clustered in a large operon termed the coboperon. A plasmid containing the following 20 genes from the cob operon(pAR8827) was transformed into E. coli and conferred the ability tosynthesize cobalamin de novo (Raux et al., J. Bacteriol. 178:753-767(1996)). To further improve yield of the cobyric acid precursor, theauthors removed the known regulatory elements of cbiA and altered theRBS. The genes and corresponding GenBank identifiers and gi numbers arelisted below.

TABLE 44 Protein GenBank ID GI Number Organism cysG NP_462380.1 16766765Salmonella typhimurium cbiK NP_460970.1 16765355 Salmonella typhimuriumcbiL NP_460969.1 16765354 Salmonella typhimurium cbiH NP_460972.116765357 Salmonella typhimurium cbiF NP_460974.1 16765359 Salmonellatyphimurium cbiG NP_460973.1 16765358 Salmonella typhimurium cbiDNP_460977.1 16765362 Salmonella typhimurium cbiJ NP_460971.1 16765356Salmonella typhimurium cbiE NP_460976.1 16765361 Salmonella typhimuriumcbiT NP_460975.1 16765360 Salmonella typhimurium cbiC NP_460978.116765363 Salmonella typhimurium cbiA NP_460980.1 16765365 Salmonellatyphimurium fldA NP_459679.1 16764064 Salmonella typhimurium cobAP31570.1 399274 Salmonella typhimurium cbiP AAA27268.1 154436 Salmonellatyphimurium cbiB Q05600.1 543942 Salmonella typhimurium cobU NP_460963.116765348 Salmonella typhimurium cobT NP_460961.1 16765346 Salmonellatyphimurium cobs AAA27270.1 154438 Salmonella typhimurium cobCNP_459635.1 16764020 Salmonella typhimurium cysG NP_462380.1 16766765Salmonella typhimurium

Some organisms unable to synthesize B₁₂ de novo are able to catalyzesome steps of the pathway. E coli, for example, is unable to synthesizethe corrin ring structure but encodes proteins that catalyze severalreactions in the pathway (Raux et al., J. Bacteriol. 178:753-767(1996)). The cysG gene encodes a functional CysG, a multifunctionalenzyme that converts uroporphyrinogen III to precorrin-2 (Hugler et al.,J. Bacteriol. 184:2404-2410 (2002); Ishige et al., Appl. Environ.Microbiol. 68:1192-1195 (2002)). The proteins encoded by cobTSUtransform cobinamide to cobalamin and introduce the 5′-deoxyadenosylgroup (Raux et al., J. Bacteriol. 178:753-767 (1996)).

TABLE 45 GI Protein GenBank ID Number Organism cobT NP_416495.1 16129932Escherichia coli K12 sp. MG1655 cobS NP_416496.1 16129933 Escherichiacoli K12 sp. MG1655 cobU NP_416497.1 16129934 Escherichia coli K12 sp.MG1655 cysG NP_417827.1 16131246 Escherichia coli K12 sp. MG1655

S. cerevisiae is not able to synthesize B₁₂ de novo, nor is it able touptake the vitamin at detectable levels. However, the S. cerevisiaegenome encodes two proteins, Met1p and Met8p, that catalyze several B₁₂pathway reactions. Met1p is analogous to the uroporphyrinogen IIItransmethylase CysG of S. typhimurium, which catalyzes the first step ofB 12 biosynthesis from uroporphyrinogen 111 (Raux et al., Biochem. J.338 (Pt 3):701-708 (1999)). The Met8p protein is a bifunctional proteinwith uroporphyrinogen III transmethylase activity and cobaltochelataseactivity analogous to the CysG of B. megaterium (Raux et al., Biochem.J. 338 (Pt 3):701-708 (1999)).

TABLE 46 Protein GenBank ID GI Number Organism Met1p NP_012995.1 6322922Saccharomyces cerevisiae Met8p NP_009772.1 6319690 Saccharomycescerevisiae

Any or all of these genes can be introduced into an organism deficientor inefficient in one or more components of cobalamin synthesis toenable or increase the efficiency of cobalamin synthesis.

Example V Engineering Enhanced Cobalamin Uptake Capability in anOrganism

This example describes engineering B₁₂ uptake capability into a hostorganism. B₁₂ uptake requires a specific transport system (Sennett etal., Annu. Rev. Biochem. 50:1053-1086 (1981)). The B₁₂ transport systemof E. coli has been extensively studied. High-affinity transport acrossthe outer membrane is calcium-dependent and mediated by a 66 kDa outermembrane porin, BtuB (Heller et al., J. Bacteriol. 161:896-903 (1985)).BtuB interacts with the TonB energy transducing system (TonB-ExbB-ExbD),facilitating energy-dependent translocation and binding to periplasmicbinding protein BtuF (Heller et al., J. Bacteriol. 161:896-903 (1985);Raux et al., J. Bacteriol. 178:753-767 (1996)). Transport across theinner membrane is facilitated by an ABC type uptake system composed ofBtuF, BtuD (ATP binding component) and BtuC (permease) (Raux et al.,Biochem. J. 338 (Pt 3):701-708 (1999)). Crystal structures of the BtuCDFcomplex are available (Raux et al., J. Bacteriol. 178:753-767 (1996);Raux et al., Biochem. J. 338 (Pt 3):701-708 (1999)). An additionalprotein, BtuE, is coexpressed in the btuCED operon, but this protein isnot required for B 12 transport and its function is unknown (Rioux andKadner, Mol. Gen. Genet. 217:301-308.(1989)). The btuCED operon isconstitutively expressed. The GenBank identifiers and GI numbers of thegenes associated with B₁₂ transport are listed below.

TABLE 47 GI Protein GenBank ID Number Organism btuB NP_418401.1 16131804Escherichia coli K12 sp. MG1655 btuC NP_416226.1 16129667 Escherichiacoli K12 sp. MG1655 btuD NP_416224.1 16129665 Escherichia coli K12 sp.MG1655 btuF NP_414700.1 16128151 Escherichia coli K12 sp. MG1655 tonBNP_415768.1 16129213 Escherichia coli K12 sp. MG1655 exbB NP_417479.116130904 Escherichia coli K12 sp. MG1655 exbD NP_417478.1 16130903Escherichia coli K12 sp. MG1655

The B₁₂ uptake capability of an organism can be further improved byoverexpressing genes encoding the requisite transport proteins, andreducing or eliminating negative regulatory control. Overexpres sing thebtuBCDF genes leads to increased binding of B12 to membranes andincreased rate of uptake into cells. Another strategy is to removeregulatory control. The btuB mRNA translation is directly repressed byB12 at the 5′ UTR (Nahvi et al., Chem. Biol. 9:1043 (2002)). Thisinteraction may induce mRNA folding to block ribosome access to thetranslational start. Mutation or elimination of the B₁₂ binding siteremoves inhibition and improves the efficiency of B₁₂ uptake (Bulthuiset al., U.S. Pat. No. 6,432,686). These strategies were successfullyemployed to improve B₁₂ uptake capability in 1,3-PDO producingmicroorganisms (WO/1999/058686) and (Bulthuis et al., U.S. Pat. No.6,432,686). A recent patent application describes improving theefficiency of B₁₂ uptake (WO/2008/152016) by deleting negativeregulatory proteins such as C. glutamicum btuR2.

S. typhimurium possesses both high and low affinity transporters forB₁₂. The high affinity transporter is encoded by btuB (Rioux and Kadner,J. Bacteriol. 171:2986-2993 (1989)). Like E. coli transport across theperiplasmic membrane is predicted to occur via an ABC transport system,although this has not been characterized to date. The B₁₂ bindingprotein is encoded by btuD and btuE, and btuC is predicted to encode thepermease.

TABLE 48 Protein GenBank ID GI Number Organism btuB AAA27031.1 153891Salmonella typhimurium LT2 btuC NP_460306.1 16764691 Salmonellatyphimurium LT2 btuD NP_460308.1 16764693 Salmonella typhimurium LT2btuE AAL20266.1 16419860 Salmonella typhimurium LT2

Any or all of these genes can be introduced into an organism deficientin one or more components of cobalamin uptake to enable or increase theefficienty cobalamin uptake.

Method for quantifying B₁₂ in the culture medium. To quantify the amountof B₁₂ in the culture medium, cell free samples are run on HPLC.Cobalamin quantification is achieved by comparing peak area ratios at278 nm and 361 num with standards, then applying peak areas to standardcurves of cobalamin.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples and studies detailed above are onlyillustrative of the invention. It should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A non-naturally occurring microbial organism, comprising a microbialorganism having an isopropanol pathway comprising at least one exogenousnucleic acid encoding an isopropanol pathway enzyme expressed in asufficient amount to produce isopropanol, said isopropanol pathwayenzyme comprising a succinyl-CoA:3-ketoacid-CoA transferase, anacetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, aphosphotransacetoacetylase, and an acetoacetate kinase.
 2. (canceled) 3.The organism of claim 1, further comprising an acetoacetyl-CoA thiolase,an acetoacetate decarboxylase, and an isopropanol dehydrogenase. 4-6.(canceled)
 7. The organism of claim 1, further comprising at least oneenzyme or polypeptide selected from the group consisting of a corrinoidprotein, a methyltetrahydrofolate:corrinoid protein methyltransferase, acorrinoid iron-sulfur protein, a nickel-protein assembly protein, aferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, apyruvate ferredoxin oxidoreductase, and a hydrogenase. 8-17. (canceled)18. The organism of claim 7, further comprising the polypetide AcsEpsencoded by the gene acsEps.
 19. The organism of claim 7, furthercomprising at least one enzyme or polypeptide encoded by a gene selectedfrom the group consisting of codh, codh-I, cooF, hypA, cooH, cooU, cooX,cooL, cooK, cooM, cooT, cooJ, and codh-II.
 20. The organism of claim 7,further comprising a methanol methyltransferase.
 21. (canceled)
 22. Theorganism of claim 20, wherein said organism utilizes a feedstockselected from the group consisting of: 1) methanol and CO, 2) methanol,CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4) methanol and synthesis gascomprising CO and H₂, 5) methanol and synthesis gas comprising CO, CO₂,and H₂, 6) one or more carbohydrates, 7) methanol and one or morecarbohydrates, and 8) methanol.
 23. The organism of claim 7, furthercomprising a formate dehydrogenase, a formyltetrahydrofolate synthetase,a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase. 24-28.(canceled)
 29. The organism of claim 23, wherein said organism utilizesa feedstock selected from the group consisting of: 1) CO, 2) CO₂ and H₂,3) CO and CO₂, 4) synthesis gas comprising CO and H₂, 5) synthesis gascomprising CO, CO₂, and H₂, and 6) one or more carbohydrates.
 30. Anon-naturally occurring microbial organism, comprising a microbialorganism having a 4-hydroxybutryate pathway comprising at least oneexogenous nucleic acid encoding an 4-hydroxybutryate pathway enzymeexpressed in a sufficient amount to produce 4-hydroxybutryate, said4-hydroxybutryate pathway enzyme comprising an acetoacetyl-CoA thiolase,a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoAhydratase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoAhydrolase, a 4-hydroxybutyryl-CoA synthetase, aphosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase. 31-37.(canceled)
 38. The organism of claim 30, further comprising at least oneenzyme or polypeptide selected from the group consisting of, a corrinoidprotein, a methyltetrahydrofolate:corrinoid protein methyltransferase, acorrinoid iron-sulfur protein, a nickel-protein assembly protein, aferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, apyruvate ferredoxin oxidoreductase, and a hydrogenase. 39-48. (canceled)49. The organism of claim 38, further comprising the polypetide AcsEpsencoded by the gene acsEps.
 50. The organism of claim 38, furthercomprising at least one enzyme or polypeptide encoded by a gene selectedfrom the group consisting of codh, codh-I, cooF, hypA, cooH, cooU, cooX,cooL, cooK, cooM, cooT, cooJ, and codh-II.
 51. The organism of claim 38,further comprising a methanol methyltransferase.
 52. (canceled)
 53. Theorganism of claim 51, wherein said organism utilizes a feedstockselected from the group consisting of: 1) methanol and CO, 2) methanol,CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4) methanol and synthesis gascomprising CO and H₂, 5) methanol and synthesis gas comprising CO, CO₂,and H₂, 6) one or more carbohydrates, 7) methanol and one or morecarbohydrates, and 8) methanol.
 54. The organism of claim 38, furthercomprising a formate dehydrogenase, a formyltetrahydrofolate synthetase,a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase. 55-59.(canceled)
 60. The organism of claim 54, wherein said organism utilizesa feedstock selected from the group consisting of: 1) CO, 2) CO₂ and H₂,3) CO and CO₂, 4) synthesis gas comprising CO and H₂, 5) synthesis gascomprising CO, CO₂, and H₂, and 6) one or more carbohydrates.
 61. Anon-naturally occurring microbial organism comprising a microbialorganism having a 1,4-butanediol pathway comprising at least oneexogenous nucleic acid encoding a 1,4-butanediol pathway enzymeexpressed in a sufficient amount to produce 1,4-butanediol, said1,4-butanediol pathway enzyme comprising an acetoacetyl-CoA thiolase, a3-Hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoAhydratase, a 4-hydroxybutyryl-CoA reductase (alcohol forming), a4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanedioldehydrogenase; said organism further comprising an acetyl-CoA pathwaycomprising at least one exogenous nucleic acid encoding an acetyl-CoApathway enzyme expressed in a sufficient amount to produce acetyl-CoA,said acetyl-CoA pathway enzyme comprising a corrinoid protein, amethyltetrahydrofolate:corrinoid protein methyltransferase, a corrinoidiron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, anacetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvateferredoxin oxidoreductase, and a hydrogenase. 62-78. (canceled)
 79. Theorganism of claim 61, further comprising the polypetide AcsEps encodedby the gene acsEps.
 80. The organism of claim 61, further comprising atleast one enzyme or polypeptide encoded by a gene selected from thegroup consisting of codh, codh-I, cooF, hypA, cooH, cooU, cooX, cooL,cooK, cooM, cooT, cooJ, and codh-II.
 81. The organism of claim 61,further comprising a methanol methyltransferase.
 82. (canceled)
 83. Theorganism of claim 81, wherein said organism utilizes a feedstockselected from the group consisting of: 1) methanol and CO, 2) methanol,CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4) methanol and synthesis gascomprising CO and H₂, 5) methanol and synthesis gas comprising CO, CO₂,and H₂, 6) one or more carbohydrates, 7) methanol and one or morecarbohydrates, and 8) methanol.
 84. A non-naturally occurring microbialorganism comprising a microbial organism having a 1,4-butanediol pathwaycomprising at least one exogenous nucleic acid encoding a 1,4-butanediolpathway enzyme expressed in a sufficient amount to produce1,4-butanediol, said 1,4-butanediol pathway enzyme comprising anacetoacetyl-CoA thiolase, a 3-Hydroxybutyryl-CoA dehydrogenase, acrotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA reductase(alcohol forming), a 4-hydroxybutyryl-CoA reductase (aldehyde forming),and a 1,4-butanediol dehydrogenase; said organism further comprising anacetyl-CoA pathway comprising at least one exogenous nucleic acidencoding an acetyl-CoA pathway enzyme expressed in a sufficient amountto produce acetyl-CoA, said acetyl-CoA pathway enzyme comprising anacetyl-CoA synthase, a formate dehydrogenase, a formyltetrahydrofolatesynthetase, a methenyltetrahydrofolate cyclohydrolase, amethylenetetrahydrofolate dehydrogenase, and a methylenetetrahydrofolatereductase. 85-89. (canceled)
 90. The organism of claim 84, wherein saidorganism utilizes a feedstock selected from the group consisting of: 1)CO, 2) CO₂ and H₂, 3) CO and CO₂, 4) synthesis gas comprising CO and H₂,5) synthesis gas comprising CO, CO₂, and H₂, and 6) one or morecarbohydrates.
 91. A non-naturally occurring microbial organismcomprising a microbial organism having an isopropanol pathway comprisingat least one exogenous nucleic acid encoding an isopropanol pathwayenzyme expressed in a sufficient amount to produce isopropanol, saidisopropanol pathway enzyme comprising an acetoacetyl-CoA thiolase, anacetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, anacetoacetyl-CoA synthetase, a phosphotransacetoacetylase, anacetoacetate kinase, an acetoacetate decarboxylase, and an isopropanoldehydrogenase; said organism further comprising at least one exogenousnucleic acid encoding an acetyl-CoA enzyme expressed in a sufficientamount to produce acetyl-CoA, said acetyl-CoA pathway enzyme comprisinga methanol methyl transferase, a corrinoid protein, amethyltetrahydro-folate:corrinoid protein methyltransferase, a corrinoidiron-sulfur protein, a nickel-protein assembly protein, a ferredoxin, anacetyl-CoA synthase, a carbon monoxide dehydrogenase, a pyruvateferredoxin oxidoreductase, and a hydrogenase.
 92. (canceled)
 93. Anon-naturally occurring microbial organism, comprising a microbialorganism having an isopropanol pathway comprising at least one exogenousnucleic acid encoding an isopropanol pathway enzyme expressed in asufficient amount to produce isopropanol, said isopropanol pathwayenzyme comprising an acetoacetyl-CoA thiolase, an acetoacetyl-CoAtransferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoAsynthetase, a phosphotransacetoacetylase, an acetoacetate kinase, anacetoacetate decarboxylase, and an isopropanol dehydrogenase; saidorganism further comprising at least one exogenous nucleic acid encodingan acetyl-CoA enzyme expressed in a sufficient amount to produceacetyl-CoA, said acetyl-CoA pathway enzyme comprising an acetyl-CoAsynthase, a formate dehydrogenase, a formyltetrahydrofolate synthetase,a methenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase.
 94. (canceled)95. A method for producing isopropanol, comprising culturing anon-naturally occurring microbial organism according to claim 1 underconditions and for a sufficient period of time to produce isopropanol.96. (canceled)
 97. The method of claim 95, further comprising anacetoacetyl-CoA thiolase, an acetoacetate decarboxylase, and anisopropanol dehydrogenase. 98-100. (canceled)
 101. The method of claim95, wherein said organism further comprises at least one enzyme orpolypeptide selected from the group consisting of, a corrinoid protein,a methyltetrahydrofolate:corrinoid protein methyltransferase, acorrinoid iron-sulfur protein, a nickel-protein assembly protein, aferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, apyruvate ferredoxin oxidoreductase, and a hydrogenase. 102-111.(canceled)
 112. The method of claim 101, further comprising thepolypetide AcsEps encoded by the gene acsEps.
 113. The method of claim101, further comprising at least one enzyme or polypeptide encoded by agene selected from the group consisting of codh, codh-I, cooF, hypA,cooH, cooU, cooX, cooL, cooK, cooM, cooT, cooJ, and codh-II.
 114. Themethod of claim 101, further comprising a methanol methyltransferase.115. (canceled)
 116. The method of claim 114, wherein said organismutilizes a feedstock selected from the group consisting of: 1) methanoland CO, 2) methanol, CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4)methanol and synthesis gas comprising CO and H₂, 5) methanol andsynthesis gas comprising CO, CO₂, and H₂, 6) one or more carbohydrates,7) methanol and one or more carbohydrates, and 8) methanol.
 117. Themethod of claim 101, wherein said organism further comprises a formatedehydrogenase, a formyltetrahydrofolate synthetase, amethenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase. 118-122.(canceled)
 123. The method of claim 117, wherein said organism utilizesa feedstock selected from the group consisting of: 1) CO, 2) CO₂ and H₂,3) CO and CO₂, 4) synthesis gas comprising CO and H₂, 5) synthesis gascomprising CO, CO₂, and H₂, and 6) one or more carbohydrates.
 124. Amethod for producing 4-hydroxybutyrate, comprising culturing anon-naturally occurring microbial organism according to claim 30 underconditions and for a sufficient period of time to produce4-hydroxybutyrate. 125-131. (canceled)
 132. The method of claim 124,wherein said organism further comprises at least one enzyme orpolypeptide selected from the group consisting of, a corrinoid protein,a methyltetrahydrofolate:corrinoid protein methyltransferase, acorrinoid iron-sulfur protein, a nickel-protein assembly protein, aferredoxin, an acetyl-CoA synthase, a carbon monoxide dehydrogenase, apyruvate ferredoxin oxidoreductase, and a hydrogenase. 133-142.(canceled)
 143. The method of claim 132, further comprising thepolypetide AcsEps encoded by the gene acsEps.
 144. The method of claim132, further comprising at least one enzyme or polypeptide encoded by agene selected from the group consisting of codh, codh-I, cooF, hypA,cooH, cooU, cooX, cooL, cooK, cooM, cooT, cooJ, and codh-II.
 145. Themethod of claim 132, further comprising a methanol methyltransferase.146. (canceled)
 147. The method of claim 145, wherein said organismutilizes a feedstock selected from the group consisting of: 1) methanoland CO, 2) methanol, CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4)methanol and synthesis gas comprising CO and H₂, 5) methanol andsynthesis gas comprising CO, CO₂, and H₂, 6) one or more carbohydrates,7) methanol and one or more carbohydrates, and 8) methanol.
 148. Themethod of claim 132, wherein said organism further comprises a formatedehydrogenase, a formyltetrahydrofolate synthetase, amethenyltetrahydrofolate cyclohydrolase, a methylenetetrahydrofolatedehydrogenase, and a methylenetetrahydrofolate reductase. 149-153.(canceled)
 154. The method of claim 148, wherein said organism utilizesa feedstock selected from the group consisting of: 1) CO, 2) CO₂ and H₂,3) CO and CO₂, 4) synthesis gas comprising CO and H₂, 5) synthesis gascomprising CO, CO₂, and H₂, and 6) one or more carbohydrates.
 155. Amethod for producing 1,4-butanediol, comprising culturing anon-naturally occurring microbial organism according to claim 61 underconditions and for a sufficient period of time to produce1,4-butanediol. 156-172. (canceled)
 173. The method of claim 155,wherein said organism further comprises the polypetide AcsEps encoded bythe gene acsEps.
 174. The method of claim 155, further comprising atleast one enzyme or polypeptide encoded by a gene selected from thegroup consisting of codh, codh-I, cooF, hypA, cooH, cooU, cooX, cooL,cooK, cooM, cooT, cooJ, and codh-II.
 175. The method of claim 155,further comprising a methanol methyltransferase.
 176. (canceled) 177.The method of claim 175, wherein said organism utilizes a feedstockselected from the group consisting of: 1) methanol and CO, 2) methanol,CO₂, and H₂, 3) methanol, CO, CO₂, and H₂, 4) methanol and synthesis gascomprising CO and H₂, 5) methanol and synthesis gas comprising CO, CO₂,and H₂, 6) one or more carbohydrates, 7) methanol and one or morecarbohydrates, and 8) methanol.
 178. A method for producing1,4-butanediol, comprising culturing a non-naturally occurring microbialorganism according to claim 84 under conditions and for a sufficientperiod of time to produce 1,4-butanediol. 179-183. (canceled)
 184. Themethod of claim 178, wherein said organism utilizes a feedstock selectedfrom the group consisting of: 1) CO, 2) CO₂ and H₂, 3) CO and CO₂, 4)synthesis gas comprising CO and H₂, 5) synthesis gas comprising CO, CO₂,and H₂, and 6) one or more carbohydrates.